Construction of fully-deleted adenovirus-based gene delivery vectors and uses thereof

ABSTRACT

The embodiments disclosed herein relate to the construction of fully-deleted Adenovirus-based gene delivery vectors packaged without helper Adenovirus, and more particularly to their use in gene therapy for gene and protein expression, vaccine development, and immunosuppressive therapy for allogeneic transplantation. In an embodiment, a method for propagating an adenoviral vector includes (a) providing an Adenovirus packaging cell line; (b) transfecting a fully-deleted Adenoviral vector construct into the cell line; and optionally (c) transfecting a packaging construct into the cell line, wherein the fully-deleted Adenoviral vector construct and optionally the packaging construct can transfect the Adenovirus packaging cell line resulting in the encapsidation of a fully-deleted Adenoviral vector independent of helper Adenovirus. In an embodiment, a target cell is transduced with the encapsidated fully-deleted Adenoviral vector for treating a condition, disease or a disorder.

RELATED APPLICATIONS

This application is a continuation of U.S. Utility application Ser. No.12/561,966, filed Sep. 17, 2009, which claims the benefit of andpriority to U.S. Provisional Application Ser. No. 61/097,735, filed Sep.17, 2008, U.S. Provisional Application Ser. No. 61/143,281, filed Jan.8, 2009, and U.S. Provisional Application Ser. No. 61/236,577, filedAug. 25, 2009, the entirety of these applications are herebyincorporated herein by reference.

FIELD

The embodiments disclosed herein relate to the construction offully-deleted Adenovirus-based gene delivery vectors packaged withouthelper Adenovirus, and more particularly to their use in gene therapyfor gene and protein expression, vaccine development andimmunosuppressive therapy.

BACKGROUND

Adenoviruses

Among the most commonly used vectors for the delivery of geneticmaterial into human cells are the Adenoviruses. Adenoviruses have beenisolated from a large number of different species, and more than 100different serotypes have been reported. The overall organization of theAdenoviral genome is conserved among serotypes, such that specificfunctions are similarly positioned. The Ad2 and Ad5 genomes have beencompletely sequenced and sequences of selected regions of genomes fromother serotypes are available. Most adults have been exposed to theAdenovirus serotypes most commonly used in gene therapy (serotypes 2 and5).

The Ad5 genome is a linear, non-segmented, double stranded DNA,approximately 34-43 kbp (size varies from group to group) which has thetheoretical capacity to encode 30-40 genes. The Ad5 genome is flanked onboth sides by inverted terminal repeat sequences (LITR and RITR), whichare essential to replication of Adenoviruses. The virus infectious cycleis divided into an early and a late phase. In the early phase, the virusis uncoated and the genome transported to the nucleus, after which theearly gene regions E1-E4 become transcriptionally active.

The early region-1 (E1) contains two transcription regions named E1A andE1B. The E1A region (sometimes referred to as immediate early region)encodes two major proteins that are involved in modification of thehost-cell cycle and activation of the other viral transcription regions.The E1B region encodes two major proteins, 19K and 55K, that prevent,via different routes, the induction of apoptosis resulting from theactivity of the E1A proteins. In addition, the E1B-55K protein isrequired in the late phase for selective viral mRNA transport andinhibition of host protein expression. Early region-2 (E2) is alsodivided into an E2A and E2B region that together encode three proteins,DNA binding protein, viral polymerase and pre-terminal protein, allinvolved in replication of the viral genome. The E3 region is notnecessary for replication in vitro but encodes several proteins thatsubvert the host defense mechanism towards viral infection. The E4region encodes at least six proteins involved in several distinctfunctions related to viral mRNA splicing and transport, host-cell mRNAtransport, viral and cellular transcription and transformation.

The late proteins necessary for formation of the viral capsids andpackaging of viral genomes, are all generated from the major latetranscription unit (MLTU) that becomes fully active after the onset ofviral DNA replication. A complex process of differential splicing andpolyadenylation gives rise to more than 15 mRNA species that share atripartite leader sequence. The early proteins E1B-55K and E4-Orf3 andOrf6 play a pivotal role in the regulation of late viral mRNA processingand transport from the nucleus.

Packaging of newly formed viral genomes in pre-formed capsids ismediated by at least two Adenoviral proteins, the late protein 52/55Kand an intermediate protein IVa2, through interaction with the viralpackaging signal (Ψ) located at the left end of the Ad5 genome. A secondintermediate protein, pIX, is part of the capsid and is known tostabilize the hexon-hexon interactions. In addition, pIX has beendescribed to transactivate TATA-containing promoters like the E1Apromoter and the major late promoter (MLP).

Adenovirus-Based Vectors and Adenoviral Packaging Cell Lines

Adenovirus-based vectors have been used as a means to achieve high-levelgene transfer into various cell types, as vaccine delivery vehicles, forgene transfer into allogeneic tissue transplants for gene therapy, andto express recombinant proteins in cell lines and tissues that areotherwise difficult to transfect with high efficiency. The current knownsystems for packaging Adenovirus-based vectors consist of a host celland a source of the Adenoviral late genes. The current known host celllines, including the 293, QBI, and PERC 6 cells, express only early(non-structural) Adenovirus (Ad) genes, not the Ad late (structural)genes needed for packaging. The Ad late genes have previously beenprovided either by the Ad vectors themselves or by a helper Ad virus.Recently, “gutless” Adenoviral vectors—vectors that are devoid of allviral-protein-coding DNA sequences—have been developed. The gutlessAdenoviral vectors contain only the ends of the viral genome (LITR andRITR), therapeutic gene sequences, and the normal packaging recognitionsignal (Ψ), which allows this genome to be selectively packaged andreleased from cells. However, to propogate the gutless adenoviral vectorrequires a helper adenovirus (the helper) that contains the adenoviralgenes required for replication and virion assembly as well as LITR,RITR, and Ψ. While this helper-dependent system allows the introductionof up to about 32 kb of foreign DNA, the helper virus contaminates thepreparations of gutless Adenoviral vectors. This contaminatingreplication competent helper virus poses serious problems for genetherapy, vaccine, and transplant applications both because of thereplication competent virus and because of the host's immune response tothe adenoviral genes in the helper virus. One approach to decreasehelper contamination in this helper-dependent vector system, has been tointroduce a conditional gene defect in the packaging recognition signal(Ψ) making it less likely that its DNA is packaged into a virion.Gutless Adenoviral vectors produced in such systems still havesignificant contamination with helper virus. Being able to producegutless Adenoviral gene transfer vectors without helper viruscontamination would offer further reduced toxicity and prolonged geneexpression in animals.

It is believed that these Ad late genes in the vector or in the helperAd virus: 1) contribute to the inflammatory response to the Adenovirusvector in gene therapy applications, 2) interfere with the immuneresponse in vaccine applications, 3) induce immune non-responsiveness toAdenovirus in allogeneic transplant applications, and 4) result inprotein contaminants in protein expression applications. Further, theyoccupy space in the Adenoviral vector that could beneficially be usedfor carrying other genetic information. Remarkable progress has beenmade with these vectors in the last decade, but some shortcomingscontinue to challenge investigators.

Adenovirus Vectors for Gene Therapy and Protein Expression

Gene delivery or gene therapy is a promising method for the treatment ofacquired and inherited diseases. An ever-expanding array of genes forwhich abnormal expression is associated with life-threatening humandiseases are being cloned and identified. The ability to express suchcloned genes in humans will ultimately permit the prevention and/or cureof many important human diseases, diseases for which current therapiesare either inadequate or non-existent. Unfortunately, however, genetherapy protocols described to date have been plagued by a variety ofproblems, including in particular the short period of gene expressionfrom the vector and the inability to effectively readminister the samevector a second time, both of which are caused by the host immuneresponse against antigens associated with the vector and its therapeuticpayload. Tissues that have incorporated the viral and/or therapeuticgenes are initially attacked by the host's cellular immune response,mediated by CD8+ cytotoxic T cells as well as CD4+ helper T cells, whichdramatically limits the persistence of gene expression from the vectors.Moreover, the host's humoral immune response mediated by the CD4+ Tcells further limits the effectiveness of current gene therapy protocolsby inhibiting the successful readministration of the same vector.

For example, following an initial administration of an Adenoviralvector, serotype-specific antibodies are generated against epitopes ofthe major viral capsid proteins, namely the penton, hexon and fiber.Given that such capsid proteins are the means by which the Adenovirusattaches itself to a cell and subsequently infects the cell, suchantibodies are then able to block or “neutralize” reinfection of a cellby the same serotype of Adenovirus. This necessitates using a differentserotype of Adenovirus in order to administer one or more subsequentdoses of exogenous therapeutic DNA in the context of gene therapy andvaccines. In addition, both therapeutic and viral gene products areexpressed on the target cells making them susceptible to cellular immuneresponses. Thus, they are rejected and the beneficial effect of the genetherapy is negated and the target organ or tissue may be destroyed. As aresult of these immune-related obstacles, progress in gene therapyprotocols has been stymied.

A large research effort has been mounted to optimize virus-based genetransfer vectors. Yet, the initial promise of gene therapy has beenundermined by the biology of the commonly used viral vectors. Forexample, retroviruses are intrinsically mutagenic and oncogenic as theyintegrate into the human genome, and currently available Adenoviralvectors induce vigorous humoral and cellular immune responses thatnegate their therapeutic potential. Although the mutagenic and oncogenicproperties of retroviral vectors are intrinsic, the immunogenicity ofAdenoviral vectors may be mitigated. These responses arise fromAdenoviral gene products expressed from the Adenoviral vector itself orfrom associated helper virus.

Adenovirus Vectors for Immunosuppressive Therapy

Transplants of allogeneic cells and tissues are an increasingly frequentand important method of treating various disease and conditions. Withthe advent of embryonic and other stem cell based therapies, there willbe a further increase in such transplants. One challenge faced by suchtransplants is rejection by the recipient's immune system. Suchrejection is prevented by long-term treatment with general immunesuppressants such as rapamycin and cyclosporine A. However, treatmentwith such general immune suppressants results in an inhibition ofprotective immunity, resulting in susceptibility to a host of bacterial,viral, and fungal infections with associated morbidity and mortality.

A number of methods have been proposed for inducing specific immunesuppression directed at the allogeneic cells or tissues transplanted.One of these methods is based on the classical “veto effect” thatemploys donor-derived CD8⁺ T cells to inhibit cellular immune responses.Yet, allogeneic grafts may only be partially protected by classical vetoas CD8⁺ T cells may fail to remove organ-specific allo-reactive T cells.

Inducing the veto effect can be accomplished by a number of methods thatresult in the presence of CD8 on the surface of the transplantedallogeneic cells, including treating the allogeneic transplant with aprotein fusion of CD8 and an antibody specific for a protein present ofthe allogeneic cells or tissues to be transplanted. The CD8 can also be“engineered” to the surface of the cells by introducing atranscriptionally and translationally active copy of the CD8 gene to thecells or tissues to be transplanted.

Adenoviral vectors are particularly suited for transduction of the CD8gene to allogeneic cells/tissues for transplantation because they infecta wide range of cell and tissues with high efficiency and because thetransduced DNA is expressed transiently and not permanently integratedinto the genome of the transduced cells.

The ability of the CD8 to induce long-term immune non-responsivenessraises a challenge for the use of Adenoviral vectors: the expression ofAdenoviral genes in conjunction with the CD8 gene form cells in theallogeneic transplant may induce the transplant recipient to a state oflong-term non-responsiveness to the Adenovirus used as the basis for thevector. Adenovirus is a human pathogen and though not normally a greatrisk, it is associated with significant morbidity in immunocompromisedpeople such as AIDS patients incapable of mounting an immune response toit.

At least 53 different forms of human Adenovirus have been characterized.The discriminating factor among these viruses is the humoral immune(i.e. antibody) response to the capsid hexon protein (encoded by variousalleles of the L3 gene). In fact, the majority of variation among thedifferent hexon proteins occurs in three “hyper”-variable regions; thehumoral immune response to Adenoviruses is centered on thesehypervariable regions.

The use of Adenovirus to deliver CD8 to protect allogeneic transplantsfrom rejection poses unique problems, not the same as posed by otheruses of Adenoviral vectors for more standard gene therapy protocols.Specifically, in standard gene therapy, the injection of a large numberof Adenovirus particles into the patient may activate the pre-existingimmunity to the Adenoviral vector which can interfere with thetransduction of the therapeutic gene, lead to inflammatory responses,and in extreme cases the immune response result in death of the patient.The source of Adenoviral antigens engaging the pre-existing immunity cancome both from the gene therapy virions and from newly synthesizedAdenoviral proteins produced by infected cells.

Two advances have sought to overcome these problems are the use of“gutless” (fully-deleted) Adenoviral vectors and the use of rareAdenoviral hexons. While the use of “gutless” Adenoviral vectors removesthe L3 gene from the therapeutic vector, the propagation of these“gutless” viruses requires the presence of helper Adenovirus that stillcontains L3 genes. And these helper viruses are significant contaminantsin the therapeutic preparations of the “gutless” Adenoviral vectors. Theuse of L3 genes from rare adenoserotypes may avoid the problem ofpre-existing immunity in that fraction of patients who have not beenpreviously exposed to the Adenoviral serotype. Still, as the Adenoviralhexon proteins are highly immunogenic, there is a high probability thatrepeated treatments with an Adenoviral gene delivery vector based on arare serotype will eventually induce an immune reaction, includingneutralizing antibodies. In summary, the problem with Adenoviral vectorsfor classical gene therapy protocols is the presence or development ofan immune response to the Adenoviral proteins that interferes withtransduction of the therapeutic gene and/or causes inflammatory or otherimmune responses.

In the use of Adenovirus to deliver the CD8 gene to allogeneiccells/tissues ex vivo before reimplantation, the problem of immunereaction to the Adenoviral genes is quite different: here one isconcerned with the induction of long-term immune non-responsiveness towhichever Adenovirus serotype serves as the basis for the vector.

Adenoviruses as Vaccine Vectors

Adenoviruses have transitioned from tools for gene replacement therapyto bona fide vaccine delivery vehicles. They are attractive vaccinevectors as they induce both innate and adaptive immune responses inmammalian hosts. Currently, Adenovirus vectors are being tested assubunit vaccine systems for numerous infectious agents ranging frommalaria to HIV-1. Additionally, they are being explored as vaccinesagainst a multitude of tumor-associated antigens. Thus far, most effortshave focused on vectors derived from Adenovirus of the human serotype 5(AdHu5) for eventual use as vaccines for humans, while bovine, porcine,and ovine Adenoviruses have been explored for veterinary use.

The dynamics of Adenoviral gene expression have made the production oftrue Adenoviral packaging cell lines difficult: expression of theAdenoviral early functional transcription region (E1A) gene inducesexpression of the Adenoviral late genes (structural, immunogenic genes),which in turn kills the cell. Accordingly, a host cell thatconstitutively expresses the Adenoviral early genes cannot carry the“wild-type” Adenoviral late cistron. Previous host cells for propagatingAdenoviral vectors are not “packaging” cells. Specifically, the 293, QBIand PERC 6 cells express only early (non-structural) Adenoviral genes,not the Adenoviral late genes needed for packaging. The Adenoviral lategenes have previously been provided either by the Adenoviral vector orby a helper Adenoviral virus. These Adenoviral late genes in theAdenoviral vector or in a helper Adenoviral virus contribute to theinflammatory response to the Adenoviral vector; interfere with theimmune response to Adenoviral based vaccines; induce immunenon-responsiveness to Adenovirus in allogeneic transplant applications,and contribute to contamination in Adenoviral based protein expression.Further, they occupy space that could beneficially be used for carryingother genetic information.

The described invention addresses this problem and provides systems andmethods for the construction of fully-deleted helper-independentAdenoviral vectors and uses thereof.

SUMMARY

The embodiments disclosed herein relate to the construction offully-deleted Adenovirus-based gene transfer vectors (GDVs) packagedwithout the use of helper Adenovirus, and more particularly to their usein gene therapy for gene and protein expression, vaccine development,and immunosuppressive therapy.

According to aspects illustrated herein, there is provided an Adenoviruspackaging cell line permissive for replication of a fully-deletedAdenoviral vector independent of helper Adenovirus that includes anAdenovirus early region 1 (E1) coding sequence and an Adenovirus pIXcoding sequence, both of which are stably integrated into the cell line.In a preferred embodiment, no additional viral coding sequences arepresent in the Adenovirus packaging cell line.

According to aspects illustrated herein, there is provided a system thatincludes (a) an Adenovirus packaging cell line; (b) a fully-deletedAdenoviral vector construct; and optionally (c) a packaging construct,wherein the fully-deleted Adenoviral vector construct and optionally thepackaging construct can transfect the Adenovirus packaging cell lineresulting in the encapsidation of a fully-deleted Adenoviral vectorindependent of helper Adenovirus. The packaging construct itself isincapable of being packaged, and the encapsidated fully-deletedAdenoviral vector is replication deficient.

According to aspects illustrated herein, there is disclosed a method forproducing an Adenovirus packaging cell line permissive for replicationof a fully-deleted Adenoviral vector independent of helper Adenovirusthat includes introducing into a cell line permissive for Adenovirusreplication an isolated nucleic acid molecule comprising an Adenovirusearly region 1 (E1) coding sequence and an Adenovirus pIX codingsequence, wherein the Adenovirus early region 1 (E1) coding sequence andthe Adenovirus pIX coding sequence are stably integrated into the cellline.

According to aspects illustrated herein, there is disclosed a method forpropagating an adenoviral vector that includes (a) providing anAdenovirus packaging cell line; (b) transfecting a fully-deletedAdenoviral vector construct into the cell line; and optionally (c)transfecting a packaging construct into the cell line, wherein thefully-deleted Adenoviral vector construct and optionally the packagingconstruct can transfect the Adenovirus packaging cell line resulting inthe encapsidation of a fully-deleted Adenoviral vector independent ofhelper Adenovirus.

In some embodiments, gene transfer vectors (GTVs) of the presentdisclosure are useful in a method of treating cancer. In someembodiments, gene transfer vectors (GTVs) of the present disclosure areuseful in a method of treating skin disorders. In some embodiments, genetransfer vectors (GTVs) of the present disclosure are useful in a methodof treating vascular disease. In some embodiments, gene transfer vectors(GTVs) of the present disclosure are useful in a method of treatingcardiac disease. In some embodiments, gene transfer vectors (GTVs) ofthe present disclosure are useful in a method of treating an auto-immunedisease. In some embodiments, gene transfer vectors (GTVs) of thepresent disclosure are useful in a method of treating a parasiticinfection. In some embodiments, gene transfer vectors (GTVs) of thepresent disclosure are useful in a method of treating a viral infection.In some embodiments, gene transfer vectors (GTVs) of the presentdisclosure are useful in a method of treating a bacterial infection. Insome embodiments, gene transfer vectors (GTVs) of the present disclosureare useful in a method of treating a yeast infection. In someembodiments, gene transfer vectors (GTVs) of the present disclosure areuseful in a method of treating a neurological disease. In someembodiments, gene transfer vectors (GTVs) of the present disclosure areuseful in a method of treating a hereditary disease.

In some embodiments, an encapsidated fully-deleted Adenoviral vectorproduced by a method of the present disclosure is used as a genedelivery vector for protein expression. In some embodiments, anencapsidated fully-deleted Adenoviral vector produced by a method of thepresent disclosure is used in developing and manufacturing vaccines. Insome embodiments, an encapsidated fully-deleted Adenoviral vectorproduced by a method of the present disclosure is used as a genedelivery vector for immunosuppressive therapy. In an embodiment, atarget cell is transduced with an encapsidated fully-deleted Adenoviralvector produced by a method of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation showing an embodiment of amethod for the creation of a fully-deleted Adenoviral vector construct(FDV) using homologous recombination with a plasmid comprising stufferDNA.

FIG. 2 is a set of schematic diagrams showing four embodiments offully-deleted Adenoviral vector constructs (FDVs) of the presentdisclosure.

FIG. 3 is a diagrammatic representation showing an embodiment of amethod for the creation of an Adenoviral packaging construct (pPack)using wild-type Adenovirus DNA.

FIG. 4 is a set of schematic diagrams showing six embodiments ofpackaging constructs (pPacks) of the present disclosure.

FIG. 5 is a diagrammatic representation showing an embodiment of aco-transfection system comprising a fully-deleted Adenoviral vectorconstruct (FDV), a packing construct (pPack), and an Adenoviruspackaging cell (PC) for producing encapsidated fully-deletedAdenovirus-based gene transfer vectors (GDVs) without helper Adenovirus.

FIG. 6 is a diagrammatic representation showing an embodiment of atransfection system comprising a fully-deleted Adenoviral vectorconstruct (FDV) and an Adenovirus packaging cell (PC) for producingencapsidated fully-deleted Adenovirus-based gene transfer vectors (GDVs)without helper Adenovirus.

FIG. 7 is a diagrammatic representation showing an embodiment of amethod for the creation of a packing construct (pPack) expressingAdenoviral late genes. In this example, the Adenoviral late genes areexcised in portions of about 5 kb and cloned separately into plasmidvectors. The pieces of Ad DNA are then pieced back together sequentiallyinto one plasmid. Finally, the SV40 (or any non-adeno) viral early geneand origin of replication are added to the plasmid comprising there-constructed Adenovirus late region.

FIG. 8 is a diagrammatic representation showing an embodiment of amethod of producing an Adenovirus packaging cell (PC) line (mammaliancell line) expressing the E1 and IX genes from Adenovirus, permissivefor production of encapsidated fully-deleted Adenovirus-based genetransfer vectors (GDVs) without helper Adenovirus. First, the E1 and IXgenes are amplified by PCR from the wild-type Adenovirus DNA. Next, theE1/IX DNA is transfected into a human cell line (A549). Finally,transformed colonies of cells are isolated and expanded for testing.

FIG. 9 is a diagrammatic representation showing an embodiment of amethod of producing an Adenovirus packaging cell (PC) line (mammaliancell line) expressing the E1 and IX genes from Adenovirus usingco-transfection with a neomycin selection cassette, permissive forproduction of encapsidated fully-deleted Adenovirus-based gene transfervectors (GDVs) without helper Adenovirus. First, the E1 and IX genes areamplified by PCR from the wild-type Adenovirus DNA. Next, the E1/IX DNAand a neomycin selection cassette are co-transfected into a human cellline (A549). Finally, colonies resistant to G418 are isolated and testedfor expression of the E1/IX genes.

FIG. 10A-C shows the expression of Factor VIII using a GDV of thepresent invention with a DNA insert of a F8 gene. The GDV was producedby co-transfection with a packaging plasmid into QBI cells (QBiogene).The amount of GDV produced in packaging cells was measured by anAdenovirus-specific capture ELISA. mAdCD8 was used as a positive control(FIG. 10A). Fibroblasts were infected with the GDV. Factor VIII DNA wasdetected in transduced cells (virus), in mock-infected cells (neg), orin the GDV DNA (pos) by F8-specific PCR (FIG. 10B). The release of F8was detected by a F8-specific EliSpot assay in the F8 producing cells(20B8, middle panel), in mock-infected cells (neg, left panel), and inGDV-transduced fibroblasts (virus, right panel) (FIG. 10C).

DETAILED DESCRIPTION

The present disclosure provides, among other things, fully-deletedAdenoviral vector constructs (FDVs), packaging constructs (pPacks) andAdenovirus packaging cells (PCs) for propagating fully-deletedAdenovirus-based gene transfer vectors (GDVs) packaged without helperAdenovirus. The GDVs find use in gene therapy for gene and proteinexpression, vaccine development and immunosuppressive therapy. Anysubtype, mixture of subtypes, or chimeric Adenovirus may be used as thesource of DNA for generation of a FDV. In an embodiment, the source ofDNA is from Human Serotype 5. Given that the Ad5 genome has beencompletely sequenced, the present disclosure will be described withrespect to the Ad5 serotype.

To the inventors knowledge, all previous versions of Ad viral genetransfer vectors were contaminated with Adenoviral genes. In the case offirst- and second-generation Ad vectors, the Ad vector itself carriedthe Ad genes, while in the case of “gutless” Ad vectors, thecontaminating helper virus needed for production of Ad gutless vectorscarry the Ad genes. Accordingly, when cells were infected with the Adgene delivery vector they were also transduced with Ad genes. In thesystems and methods disclosed herein, the Ad genes are provided by ahost cell into which they are stably integrated and/or by a pPacknucleic acid transfected into the host cell. Accordingly, the GDVs ofthe present invention are uniquely not contaminated with Ad genes.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Generally, the nomenclatureused herein and the laboratory procedures in cell culture, moleculargenetics, and nucleic acid chemistry and hybridization described beloware those well known and commonly employed in the art. Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, and microbial culture and transformation (e.g.,electroporation, lipofection). Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference)which are provided throughout this document. Units, prefixes, andsymbols may be denoted in their SI accepted form. Unless otherwiseindicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxyl orientation, respectively. Numeric ranges are inclusive of thenumbers defining the range and include each integer within the definedrange. Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes. Unless otherwise provided for, software, electrical, andelectronics terms as used herein are as defined in The New IEEE StandardDictionary of Electrical and Electronics Terms (5^(th) edition, 1993).As employed throughout the disclosure, the following terms, unlessotherwise indicated, shall be understood to have the following meaningsand are more fully defined by reference to the specification as a whole:

The terms “Adenovirus” and “Adenoviral particle” as used herein includeany and all viruses that may be categorized as an Adenovirus, includingany Adenovirus that infects a human or an animal, including all groups,subgroups, and serotypes. Thus, as used herein, “Adenovirus” and“Adenovirus particle” refer to the virus itself or derivatives thereofand cover all serotypes and subtypes and both naturally occurring andrecombinant forms. In one embodiment, such Adenoviruses infect humancells. Such Adenoviruses may be wildtype or may be modified in variousways known in the art or as disclosed herein. Such modifications includemodifications to the Adenovirus genome that is packaged in the particlein order to make an infectious virus. Such modifications includedeletions known in the art, such as deletions in one or more of the E1a,E1b, E2a, E2b, E3, or E4 coding regions.

An “Adenovirus packaging cell” is a cell that is able to packageAdenoviral genomes or modified genomes to produce viral particles. Itcan provide a missing gene product or its equivalent. Thus, packagingcells can provide complementing functions for the genes deleted in anAdenoviral genome and are able to package the Adenoviral genomes intothe Adenovirus particle. The production of such particles requires thatthe genome be replicated and that those proteins necessary forassembling an infectious virus are produced. The particles also canrequire certain proteins necessary for the maturation of the viralparticle. Such proteins can be provided by a vector, a packagingconstruct or by the packaging cell. Exemplary host cells (HCs) that maybe used to make a packaging cell line according to the present inventioninclude, but are not limited to A549, HeLa, MRC5, W138, CHO cells, Verocells, human embryonic retinal cells, or any eukaryotic cells, as longas the host cells are permissive for growth of Adenovirus. Some hostcell lines include adipocytes, chondrocytes, epithelial, fibrobasts,glioblastoma, hepatocytes, keratinocytes, leukemia, lymphoblastoid,monocytes, macrophages, myoblasts, and neurons. Other cell typesinclude, but are not limited to, cells derived from primary cellcultures, e.g., human primary prostate cells, human embryonic retinalcells, human stem cells. Eukaryotic dipolid and aneuploid cell lines areincluded within the scope of the invention. The packaging cell must beone that is capable of expressing the products of the FDV and/or pPackconstructs at the appropriate level for those products in order togenerate a high titer stock of recombinant GDVs.

By “antigen” is meant a molecule which contains one or more epitopesthat will stimulate a host's immune system to make a cellularantigen-specific immune response, or a humoral antibody response. Thus,antigens include proteins, polypeptides, antigenic protein fragments,oligosaccharides, polysaccharides, and the like. Furthermore, theantigen can be derived from any known virus, bacterium, parasite,plants, protozoans, or fungus, and can be a whole organism. The termalso includes tumor antigens. Similarly, an oligonucleotide orpolynucleotide which expresses an antigen, such as in DNA immunizationapplications, is also included in the definition of antigen. Syntheticantigens are also included, for example, polyepitopes, flankingepitopes, and other recombinant or synthetically derived antigens(Bergmann et al. (1993) Eur. J. Immunol. 23:2777 2781; Bergmann et al.(1996) J. Immunol. 157:3242 3249; Suhrbier, A. (1997) Immunol. and CellBiol. 75:402 408; Gardner et al. (1998) 12th World AIDS Conference,Geneva, Switzerland, Jun. 28-Jul. 3, 1998).

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus A transcription terminationsequence may be located 3′ to the coding sequence. Transcription andtranslation of coding sequences are typically regulated by “controlelements,” including, but not limited to, transcription promoters,transcription enhancer elements, Shine and Delagamo sequences,transcription termination signals, polyadenylation sequences (located 3′to the translation stop codon), sequences for optimization of initiationof translation (located 5′ to the coding sequence), and translationtermination sequences.

The term “construct” refers to at least one of a fully-deletedAdenoviral vector construct (FDV) of the present invention or apackaging construct (pPack) of the present invention.

The term “delete” or “deleted” as used herein refers to expunging,erasing, or removing.

The term “E1 region” as used herein refers to a group of genes presentin the Adenovirus genome. These genes, such as, but not limited to, E1Aand E1B, are expressed in the early phase of virus replication andactivate the expression of the other viral genes. In an embodiment, anAdenoviral packaging cell line of the present disclosure includes allcoding sequences that make up the E1 region. In an embodiment, anAdenoviral packaging cell line of the present disclosure includes somecoding sequences that make up the E1 region (for example, E1A or E1B)

As used herein, the term “E1A” refers to all gene products of theAdenovirus E1A region, including expression products of the two majorRNAs: 13S and 12S. These are translated into polypeptides of 289 and 243amino acids, respectively. These two proteins differ by 46 amino acids,which are spliced from the 12S mRNA, as described in Chow et al. (1980)Cold Spring Harb Symp Quant Biol. 44 Pt 1:401 14; and Chow et al. (1979)J. Mol. Biol. 134(2):265 303, herein specifically incorporated byreference. For the purposes of the invention, the packaging cell linemay express the 289 polypeptide, the 243 polypeptide, or both the 289and the 243 polypeptide. The term E1A is also used herein with referenceto partial and variant E1A coding sequences.

As used herein, the term “E1B” refers to all gene products of theAdenovirus E1B region, including the 3 major polypeptides, of 19 kd and55 kd. The E1B 19 kd and 55 kd proteins are important in celltransformation. For the purposes of the invention, the packaging cellline may express the 19 Kd polypeptide, the 55 Kd polypeptide, or boththe 19 and the 55 Kd polypeptide. The term E1B is also used herein withreference to partial and variant E1B coding sequences.

The term “E2” as used herein refers to a cistron with at least 3 ORFsall of which are involved in DNA replication, including a polymerase.The E2 late promoter of Adenovirus has been described, for example, bySwaminathan, S., and Thimmapaya, B. (1995) Curr. Top. Microbiol.Immunol., 199, 177-194. In the Adenoviral system, the E2 late promoter,together with the E2 early promoter, has the function of controlling theadenoviral E2 region and/or genes E2A and E2B. In this case, thesynthesis of the E2 mRNA takes places initially starting out from the E2early promoter. Approximately five to seven hours after the infection ofa cell, a switch-over to the E2 later promoter takes place.

The term “E3 region” as used herein refers to a group of genes that arepresent in the Adenovirus genome and are expressed in the early phase ofthe virus replication cycle. These genes express proteins that interactwith the host immune system. They are not necessary for virusreplication in vitro, and therefore may be deleted in Adenovirusvectors.

The term “E4 region” as used herein refers to a group of genes that arepresent in the Adenovirus genome next to the right ITR, and areexpressed in the early phase of the virus replication cycle. The E4region includes at least 7 ORFs. The products of the E4 region promoteviral gene expression and replication, interact with host cellcomponents, and participate in lytic infection and oncogenesis.

The term “expression” refers to the transcription and/or translation ofan endogenous gene, transgene or coding region in a cell.

The terms “fully-deleted Adenoviral vector”, “FDV”, “gutless”, “gutted”,“mini”, “fully-deleted”, “Δ”, or “pseudo” vectors as used herein refersto a linear, double-stranded DNA molecule with inverted terminal repeats(ITRs) separated by approximately 28 to 37 kb, the viral packagingsignal (Ψ), and at least one DNA insert (all or a fragment of at leastone gene of interest (GOI)) which comprises a gene sequence encoding aprotein of interest. The gene sequence can be regulatable. Regulation ofgene expression can be accomplished by one of 1) alteration of genestructure: site-specific recombinases (e.g., Cre based on the Cre-loxPsystem) can activate gene expression by removing inserted sequencesbetween the promoter and the gene; 2) changes in transcription: eitherby induction (covered) or by relief of inhibition; 3) changes in mRNAstability, by specific sequences incorporated in the mRNA or by siRNA;and 4) changes in translation, by sequences in the mRNA. No viral codinggenes are comprised in the FDV. FDVs are also called“high-capacity”Adenoviruses because they can accommodate up to 36kilobases of DNA. As vector capsids package efficiently only DNA of75-105% of the whole Adenovirus genome, and as therapeutic expressioncassettes usually do not add up to 36 kb, there is a need to use“stuffer” DNA in order to complete the genome size for encapsidation.Conventional FDVs are referred to as “helper-dependent” Adenovirusesbecause they need a helper Adenovirus that carries essential Ad codingregions.

As used herein, the term “gene expression construct” refers to apromoter, at least a fragment of a gene of interest, and apolyadenylation signal sequence. A FDV of the present disclosurecomprises a gene expression construct.

A “gene of interest” or “GOI” can be one that exerts its effect at thelevel of RNA or protein. Examples of genes of interest include, but arenot limited to, therapeutic genes, immunomodulatory genes, virus genes,bacterial genes, protein production genes, inhibitory RNAs or proteins,and regulatory proteins. For instance, a protein encoded by atherapeutic gene can be employed in the treatment of an inheriteddisease, e.g., the use of a cDNA encoding the cystic fibrosistransmembrane conductance regulator in the treatment of cystic fibrosis.Moreover, the therapeutic gene can exert its effect at the level of RNA,for instance, by encoding an antisense message or ribozyme, an siRNA asis known in the art, an alternative RNA splice acceptor or donor, aprotein that affects splicing or 3′ processing (e.g., polyadenylation),or a protein that affects the level of expression of another gene withinthe cell (i.e., where gene expression is broadly considered to includeall steps from initiation of transcription through production of aprocessed protein), perhaps, among other things, by mediating an alteredrate of mRNA accumulation, an alteration of mRNA transport, and/or achange in post-transcriptional regulation.

As used herein, the phrase “gene therapy” refers to the transfer ofgenetic material (e.g., DNA or RNA) of interest into a host to treat orprevent a genetic or acquired disease or condition. The genetic materialof interest encodes a product (e.g., a protein polypeptide, peptide orfunctional RNA) whose production in vivo is desired. For example, thegenetic material of interest can encode a hormone, receptor, enzyme or(poly) peptide of therapeutic value. Examples of genetic material ofinterest include DNA encoding: the cystic fibrosis transmembraneregulator (CFTR), Factor VIII, low density lipoprotein receptor,betagalactosidase, alpha-galactosidase, beta-glucocerebrosidase,insulin, parathyroid hormone, and alpha-1-antitrypsin.

By “gene delivery vector” or “GDV” is meant a composition including anencapsidated fully-deleted Adenovirus-based vector of the presentdisclosure packaged without helper Adenovirus.

The term “helper-independent” as used herein refers to the process forcreating an encapsidated fully-deleted Adenovirus-based gene transfervector of the present disclosure that does not need the presence of ahelper virus for its replication. These Adenoviruses include“first-generation” and “second-generation” Adenovirus vectors. Afirst-generation Adenovirus vector refers to an Adenovirus in whichexogenous DNA replaces the E1 region, or optionally the E3 region, oroptionally both the E1 and E3 region. A second-generation Adenovirusvector refers to a first-generation Adenovirus vector, which, inaddition to the E1 and E3 regions, contains additional deletions in theE2 region, the E4 region, or any other region of the Adenovirus genome,or a combination thereof.

The term “helper virus” as used herein refers to virus used whenproducing copies of a helper-dependent viral vector which does not havethe ability to replicate on its own. The helper virus is used toco-infect cells alongside the gutless virus and provides the necessaryenzymes for replication of the genome of the gutless virus and thestructural proteins necessary for the assembly of the gutless viruscapsid.

By “immune response” is preferably meant an acquired immune response,such as a cellular or humoral immune response.

In the context of the present specification, an “immunomodulatorymolecule” is a polypeptide molecule that modulates, i.e. increases ordecreases, a cellular and/or humoral host immune response directed to atarget cell in an antigen-specific fashion, and preferably is one thatdecreases the host immune response. Generally, in accordance with theteachings of the present invention the immunomodulatory molecule(s) willbe associated with the target cell surface membrane, e.g., inserted intothe cell surface membrane or covalently or non-covalently bound thereto,after expression from the GDVs described herein. In some embodiments,the immunomodulatory molecule comprises all or a functional portion of aCD8 protein, and even more preferably all or a functional portion of theCD8α-chain. For human CD8 coding sequences, see Leahy, Faseb J. 9:17-25(1995); Leahy et al., Cell 68:1145-62 (1992); Nakayama et al.,Immunogenetics 30:393-7 (1989). By “functional portion” with respect toCD8 proteins and polypeptides is meant that portion of the CD8α-chainretaining veto activity as described herein, more particularly thatportion retaining the HLA-binding activity of the CD8α-chain, andspecifically the immunoglobulin-like domain in the extracellular regionof the CD8α-chain. Exemplary variant CD8 polypeptides are described inGao and Jakobsen, Immunology Today 21:630-636 (2000), hereinincorporated by reference. In some embodiments, the full lengthCD8α-chain is used. However, in some embodiments the cytoplasmic domainis deleted. Preferably the transmembrane domain and extracellular domainare retained.

By “immunosuppressive therapy” is meant treatment with a gene transfervector of the present invention for suppressing the immune response toantigen(s). Immunosuppressive therapy is therapy used to decrease thebody's immune response, such as drugs given to prevent or treat:transplant rejection (for example, in allogeneic transplantation), anautoimmune disease, an allergy, and a multiple myeloma.

By “inhibiting” is meant the direct or indirect, partial or complete,inhibition and/or reduction of an innate or acquired immune response,whether cellular (e.g., leukocyte recruitment) or humoral, tovector-associated antigens and/or to target cell-specific antigens.Vector-associated antigens include, e.g., antigens derived from thenucleic acid carrier or envelope (e.g. viral coat proteins and the like)as well as antigens derived from vector genes (e.g. bacterial or viralnucleic acids and proteins) and/or any therapeutic transgenes (e.g.mammalian nucleic acids and/or proteins) included in the vector.

The term “inverted terminal repeat” as used herein refers to DNAsequences located at the left and right termini of the Adenovirusgenome. These sequences are identical to each other, but placed inopposite directions. The length of the inverted terminal repeats ofAdenoviruses vary from about 50 bp to about 170 bp, depending on theserotype of the virus. The inverted terminal repeats contain a number ofdifferent cis-acting elements required for viral growth, such as thecore origin of viral DNA replication and enhancer elements for theactivation of the E1 region.

“In vivo gene therapy” and “in vitro gene therapy” are intended toencompass all past, present and future variations and modifications ofwhat is commonly known and referred to by those of ordinary skill in theart as “gene therapy”, including ex vivo applications.

The term “introducing”, as used herein refers to delivery of anexpression vector for stable integration of E1A and/or E1B codingsequences in a host cell. A vector may be introduced into the cell bytransfection, which typically means insertion of heterologous DNA into acell by physical means (e.g., calcium phosphate transfection,electroporation, microinjection or lipofection); infection, whichtypically refers to introduction by way of an infectious agent, i.e. avirus; or transduction, which typically means stable infection of a cellwith a virus or the transfer of genetic material from one microorganismto another by way of a viral agent (e.g., a bacteriophage). As set forthabove, the vector may be a plasmid, virus or other vehicle.

The term “linear DNA” as used herein refers to non-circularized DNAmolecules.

The term “naturally” as used herein refers to as found in nature;wild-type; innately or inherently.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e.g., peptide nucleic acids).

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous. However, enhancers do nothave to be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adapters or linkers are used in accordance withconventional practice.

The term “packaging construct” or “pPack” refers to an engineeredplasmid construct of circular, double-stranded DNA molecules, whereinthe DNA molecules include at least a subset of Adenoviral late genes(e.g., L1, L2, L3, L4, L5, E2A, and E4) under control of a promoter. ThepPack does not include the inverted terminal repeats (ITRs) and thepackaging signal (ψ). The pPack is “replication defective”—the viralgenome does not comprise sufficient genetic information alone to enableindependent replication to produce infectious viral particles within acell. Any subtype, mixture of subtypes, or chimeric Adenovirus may beused as the source of DNA for generation of the FDV and the pPack.However, given that the Ad5 genome has been completely sequenced, thepresent disclosure will be described with respect to the Ad5 serotype.

The term “packaging signal” as used herein refers to a nucleotidesequence that is present in the virus genome and is necessary for theincorporation of the virus genome inside the virus capsid during virusassembly. The packaging signal of Adenovirus is naturally located at theleft-end terminus, downstream from the left inverted terminal repeat. Itmay be denoted as “Ψ”.

The term “pathogen” is used in a broad sense to refer to the source ofany molecule that elicits an immune response. Thus, pathogens include,but are not limited to, virulent or attenuated viruses, bacteria, fungi,protozoa, parasites, cancer cells and the like. Typically, the immuneresponse is elicited by one or more peptides produced by thesepathogens. As described in detail below, genomic DNA encoding theantigenic peptides from these and other pathogens is used to generate animmune response that mimics the response to natural infection. It willalso be apparent in view of the teachings herein, that the methodsinclude the use of genomic DNA obtained from more than one pathogen.

A cell that is “permissive” supports replication of a virus.

The term “plasmid” as used herein refers to an extra-chromosomal DNAmolecule separate from the chromosomal DNA which is capable ofreplicating independently of the chromosomal DNA. In many cases, it iscircular and double-stranded.

The term “polylinker” is used for a short stretch of artificiallysynthesized DNA which carries a number of unique restriction sitesallowing the easy insertion of any promoter or DNA segment. The term“heterologous” is used for any combination of DNA sequences that is notnormally found intimately associated in nature.

The term “promoter” is intended to mean a regulatory region of DNA thatfacilitates the transcription of a particular gene. Promoters usuallycomprise a TATA box capable of directing RNA polymerase II to initiateRNA synthesis at the appropriate transcription initiation site for aparticular coding sequence. A promoter may additionally comprise otherrecognition sequences generally positioned upstream or 5′ to the TATAbox, referred to as upstream promoter elements, which influence thetranscription initiation rate. A “constitutive promoter” refers to apromoter that allows for continual transcription of its associated genein many cell types. An “inducible-promoter system” refers to a systemthat uses a regulating agent (including small molecules such astetracycline, peptide and steroid hormones, nerotransmitters, andenvironmental factors such as heat, and osmolarity) to induce or tosilence a gene. Such systems are “analog” in the sense that theirresponses are graduated, being dependent on the concentration of theregulating agent. Also, such systems are reversible with the withdrawalof the regulating agent. Activity of these promoters is induced by thepresence or absence of biotic or abiotic factors. Inducible promotersare a powerful tool in genetic engineering because the expression ofgenes operably linked to them can be turned on or off at certain stagesof development of an organism or in a particular tissue.

The term “propagate” or “propagated” as used herein refers to reproduce,multiply, or to increase in number, amount or extent by any process.

The term “purification” as used herein refers to the process ofpurifying or to free from foreign, extraneous, or objectionableelements.

The term “regulatory sequence” (also called “regulatory region” or“regulatory element”) as used herein refers to a promoter, enhancer orother segment of DNA where regulatory proteins such as transcriptionfactors bind preferentially. They control gene expression and thusprotein expression.

The term “recombinase” as used herein refers to an enzyme that catalyzesgenetic recombination. A recombinase enzyme catalyzes the exchange ofshort pieces of DNA between two long DNA strands, particularly theexchange of homologous regions between the paired maternal and paternalchromosomes.

The term “restriction enzyme” (or “restriction endonuclease”) refers toan enzyme that cuts double-stranded DNA.

The term “restriction sites” or “restriction recognition sites” refer toparticular sequences of nucleotides that are recognized by restrictionenzymes as sites to cut the DNA molecule. The sites are generally, butnot necessarily, palindromic, (because restriction enzymes usually bindas homodimers) and a particular enzyme may cut between two nucleotideswithin its recognition site, or somewhere nearby.

The term “replication” or “replicating” as used herein refers to makingan identical copy of an object such as, for example, but not limited to,a virus particle.

The term “replication deficient” as used herein refers to thecharacteristic of a virus that is unable to replicate in a naturalenvironment. A replication deficient virus is a virus that has beendeleted of one or more of the genes that are essential for itsreplication, such as, for example, but not limited to, the E1 genes.Replication deficient viruses can be propagated in a laboratory in celllines that express the deleted genes.

By “specific immune inhibition” or “antigen-specific immune inhibition”is meant the inhibition of immune responses directed against antigenssuch as vector-associated antigens, as opposed to general immuneinhibition which is not antigen-specific. Thus, by way of example, theabsence of a host cellular and/or humoral immune response tovector-associated antigens, combined with evidence of in vivo immunecompetence to other foreign antigens, would demonstrate specific immuneinhibition of vector-associated antigens.

By “stable immunological tolerance” is meant stable, long-term allograftsurvival and/or function for at least one year without the use ofgeneral immunosuppressive agents.

The term “stuffer” as used herein refers to a DNA sequence that isinserted into another DNA sequence in order to increase its size. Forexample, a stuffer fragment can be inserted inside the Adenovirus genometo increase its size to about 36 kb. Stuffer fragments usually do notcode for any protein nor contain regulatory elements for geneexpression, such as transcriptional enhancers or promoters.

The term “target” or “targeted” as used herein refers to a biologicalentity, such as, for example, but not limited to, a protein, cell,organ, or nucleic acid, whose activity can be modified by an externalstimulus. Depending upon the nature of the stimulus, there may be nodirect change in the target, or a conformational change in the targetmay be induced.

As used herein, a “target cell” can be present as a single entity, orcan be part of a larger collection of cells. Such a “larger collectionof cells” may comprise, for instance, a cell culture (either mixed orpure), a tissue (e.g., epithelial or other tissue), an organ (e.g.,heart, lung, liver, gallbladder, urinary bladder, eye or other organ),an organ system (e.g., circulatory system, respiratory system,gastrointestinal system, urinary system, nervous system, integumentarysystem or other organ system), or an organism (e.g., a bird, mammal,particularly a human, or the like). Preferably, the organs/tissues/cellsbeing targeted are of the circulatory system (e.g., including, but notlimited to heart, blood vessels, and blood), respiratory system (e.g.,nose, pharynx, larynx, trachea, bronchi, bronchioles, lungs, and thelike), gastrointestinal system (e.g., including mouth, pharynx,esophagus, stomach, intestines, salivary glands, pancreas, liver,gallbladder, and others), urinary system (e.g., such as kidneys,ureters, urinary bladder, urethra, and the like), nervous system (e.g.,including, but not limited to, brain and spinal cord, and special senseorgans, such as the eye) and integumentary system (e.g., skin). Evenmore preferably, the cells are selected from the group consisting ofheart, blood vessel, lung, liver, gallbladder, urinary bladder, eyecells and stem cells. In an embodiment, the target cells arehepatocytes, and a method is provided for veto vector mediatedtransplantation of allogeneic hepatocytes in a subject. In anembodiment, the target cells are keratinocytes, and a method is providedfor veto vector mediated transplantation of allogeneic keratinocytes ina subject, for example, engineered skin. In an embodiment, the targetcells are pancreatic islets. In an embodiment, the target cells arecardiomyocytes. In an embodiment, the target cells are kidney cells, anda method is provided for veto vector mediated transplantation ofallogeneic kidneys in a subject. In an embodiment, the target cells arefibroblasts, and a method is provided for veto vector mediatedtransplantation of allogeneic fibroblasts in a subject, for example,engineered skin. In an embodiment, the target cells are neurons. In anembodiment, the target cells are glia cells.

In particular, a target cell with which a GDV is contacted differs fromanother cell in that the contacted target cell comprises a particularcell-surface binding site that can be targeted by the GDV. By“particular cell-surface binding site” is meant any site (i.e., moleculeor combination of molecules) present on the surface of a cell with whichthe GDV can interact in order to attach to the cell and, thereby, enterthe cell. A particular cell-surface binding site, therefore, encompassesa cell-surface receptor and, preferably, is a protein (including amodified protein), a carbohydrate, a glycoprotein, a proteoglycan, alipid, a mucin molecule or mucoprotein, and the like. Examples ofpotential cell-surface binding sites include, but are not limited to:heparin and chondroitin sulfate moieties found on glycosaminoglycans;sialic acid moieties found on mucins, glycoproteins, and gangliosides;major histocompatability complex I (MHC I) glycoproteins; commoncarbohydrate molecules found in membrane glycoproteins, includingmannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, andgalactose; glycoproteins, such as ICAM-1, VCAM, E-selectin, P-selectin,L-selectin, and integrin molecules; and tumor-specific antigens presenton cancerous cells, such as, for instance, MUC-1 tumor-specificepitopes. However, targeting a GDV to a cell is not limited to anyspecific mechanism of cellular interaction (i.e., interaction with agiven cell-surface binding site).

The term “transfection” as used herein refers to the introduction into acell DNA as DNA (for example, introduction of an isolated nucleic acidmolecule or a construct of the present disclosure). An Adenoviralpackaging cell line disclosed herein is transfected with at least one ofa FDV or a pPack of the present disclosure. The term “transduction” asused herein refers to the introduction into a cell DNA either as DNA orby means of a GDV of the present disclosure. A GDV of the presentdisclosure can be transduced into a target cell.

The term “vector” refers to a nucleic acid used in infection of a hostcell and into which can be inserted a polynucleotide. Vectors arefrequently replicons. Expression vectors permit transcription of anucleic acid inserted therein. Some common vectors include, but are notlimited to, plasmids, cosmids, viruses, phages, recombinant expressioncassettes, and transposons. The term “vector” may also refer to anelement which aids in the transfer of a gene from one location toanother.

The term “viral DNA” as used herein refers to a sequence of DNA that isfound in virus particles.

The term “viral genome” as used herein refers to the totality of the DNAthat is found in virus particles, and that contains all the elementsnecessary for virus replication. The genome is replicated andtransmitted to the virus progeny at each cycle of virus replication.

The term “virions” as used herein refers to a viral particle. Eachvirion consists of genetic material within a protective protein capsid.

The term “wild-type” as used herein refers to the typical form of anorganism, strain, gene, protein, nucleic acid, or characteristic as itoccurs in nature. Wild-type refers to the most common phenotype in thenatural population. The terms “wild-type” and “naturally occurring” areused interchangeably.

Fully-Deleted Adenoviral Vectors (FDVs)

FDVs only carry the cis acting sequences (i.e. ITRs, ψ) necessary forviral genome replication and encapsidation. Conventional systems andmethods for packaging FDVs require their co-infection with a “helper” Advirus, which can be a source of immunogenic Ad antigens. Methods havebeen proposed to remove the contaminating Ad helper virus from thetherapeutic Ad vector preparations. One example is to flank (flox) thepackaging site ψ in the Ad-“helper” virus with lox sequences for the Crerecombinase. In theory, passage of the fully deleted Ad vector and the“floxed” Ad helper virus would decrease contamination by excising the ψ(packaging) sequence from the Ad “helper” virus and thereby preventingthe packaging of the “helper” virus. In practice, this approach has notbeen able to reduce “helper” virus contamination below 1-in-10³.

FIG. 1 shows the creation of a fully-deleted Adenovirus vector construct(FDV) of the present disclosure using homologous recombination with aplasmid comprising stuffer DNA. First, a targetting construct isengineered to comprise a gene of interest (GOI) as an expressionconstruct (including a promoter and a polyadenylation signal sequence),the LITR Ψ and RITR from Adenovirus, and two regions of homology withthe stuffer plasmid DNA sequence (left arm and right arm). The targetingconstruct and the stuffer plasmid homologously recombine in bacterialcells to produce a circular plasmid DNA comprising the plasmid backbonefrom the targeting construct and the stuffer DNA between the tworecombination sites. Upon digestion with a restriction endonuclease atthe outer edges of the LITR and the RITR (arrows), the fully-deletedAdenovirus vector construct (FDV) is produced.

In an embodiment, the GOI is a therapeutic gene, immunomodulatory gene,a vaccine gene or combinations thereof. In an embodiment, a FDV of thepresent disclosure expresses an immunomodulatory gene. In an embodiment,the immunomodulatory gene is a gene encoding human CD8. In anembodiment, a FDV of the present disclosure expresses a therapeuticgene. In an embodiment, the therapeutic gene is an interleukin. In anembodiment, the therapeutic gene is factor VIII. In some embodiments ofthe present disclosure, a FDV comprises one or more transgenes encodingtherapeutic molecules of interest along with a CD8 polypeptide describedherein. In an embodiment, the stuffer DNA of a FDV of the presentdisclosure is genomic DNA from the human adenylosuccinate lyase (ADSL)gene, which however, does not encode a functional enzyme in the purinebiosynthesis pathway and purine nucleotide cycle. The ADSL gene isexpressed constitutively as a “housekeeping” gene in most tissues. TheADSL human genomic DNA is used in the reverse complement orientation as“stuffer” DNA without the 5′ upstream sequence or first exon of the ADSLgene to prevent any transcripts from being made from this DNA. In someembodiments, a FDV of the present disclosure further includes at leastsome Adenoviral late gene sequences. In some embodiments, a FDV of thepresent disclosure further includes at least some Adenoviral late genesequences and a transcriptional activator. In some embodiments, a FDV ofthe present disclosure further includes a transcriptional activator. Inan embodiment, the transcriptional activator is a mutant TetR-VP16fusion protein. In an embodiment, the transcriptional activator is anytranscription factor not normally expressed in the host cell.

FIG. 2 is a set of schematic diagrams of four embodiments of FDVs of thepresent disclosure (i.e., FDV1, FDV2, FDV3 and FDV4), and Table 1 liststhe sequences in each FDV embodiment. All embodiments of the FDVscomprise LITR, Ψ, and RITR from Adenovirus, at least one gene expressionconstruct (with a promoter, at least a fragment of a gene of interest,and a polyadenylation signal sequence), and human genomic stuffer DNA.In some embodiments, a FDV of the present disclosure is described as anisolated nucleic acid molecule. In an embodiment, the promoter is theconstitutive cytomegalovirus (CMV) major immediate early (IE) promoter.The FDV may also comprise a subset of Adenovirus late genes (forexample, FDV2 & FDV4) and/or a transcriptional activator (for example,FDV3 & FDV4). In an embodiment, the subset of Adenovirus late genes isunder the control of one or more promoters. In an embodiment, theAdenovirus late genes (and subset of Adenovirus late genes) is chosenfrom one of L1, L2, L3, L4, L5, E2A, and E4.

TABLE 1 FDVs FDV1 Ad Backbone (LITRΨ, RITR), gene expression construct,Stuffer FDV2 Ad Backbone (LITRΨ, RITR), Subset of Adenovirus Late genes,gene expression construct, Stuffer FDV3 Ad Backbone (LITRΨ, RITR),Transcriptional Activator, gene expression construct, Stuffer FDV4 AdBackbone (LITRΨ, RITR), Subset of Adenovirus Late genes, TranscriptionalActivator, gene expression construct, StufferPackaging Constructs (pPack)

FIG. 3 shows an embodiment of the creation of an Adenoviral packagingconstruct (pPack) using wild-type Adenovirus DNA (also described inExample 2). A targeting construct is engineered within a plasmidbackbone comprising a non-Adenoviral viral early gene and origin ofreplication (ori), and two regions of homology to the Adenovirus DNAsequence located on either side of the Adenovirus late genes. In anembodiment, the viral origin of replication is SV40 and the viral earlygene is SV40 T Ag. The targeting construct and the Ad DNA areco-transformed into bacteria (the bacterial ori-amp sequence of pBR322)in which they undergo homologous recombination, producing a circularplasmid comprising the Ad late genes and a non-adeno viral Early geneand origin of replication. FIG. 4 is a set of schematic diagrams oftwelve embodiments of pPacks for use in a co-transfection system of thepresent disclosure (i.e., pPack1, pPack2, pPack3, pPack4, pPack5,pPack6, pPack7, pPack8, pPack9, pPack10, pPack11 and pPack12). ThepPacks all comprise either all of the Adenoviral late genes or a subsetthereof under control of at least one promoter. In an embodiment, theAdenovirus late genes (and subset of Adenovirus late genes) is chosenfrom one of L1, L2, L3, L4, L5, E2A, and E4. In some embodiments, apPack of the present disclosure is described as an isolated nucleic acidmolecule.

TABLE 2 pPack Ppack1 Adenovirus Late genes + gene IX + the bacterialori-amp sequence of pBR322 Ppack2 Adenovirus Late genes + the bacterialori-amp sequence of pBR322 Ppack3 Subset of Adenovirus Late genes + geneIX + the bacterial ori-amp sequence of pBR322 Ppack4 Subset ofAdenovirus Late genes + the bacterial ori-amp sequence of pBR322 Ppack5Adenovirus Late genes + gene IX + Viral Origin + Viral Early gene + thebacterial ori-amp sequence of pBR322 Ppack6 Adenovirus Late genes +Viral Origin + Viral Early gene + the bacterial ori-amp sequence ofpBR322 Ppack7 Subset of Adenovirus Late genes + gene IX + Viral Origin +Viral Early gene + the bacterial ori-amp sequence of pBR322 Ppack8Subset of Adenovirus Late genes + Viral Origin + Viral Early gene + thebacterial ori-amp sequence of pBR322 Ppack9 Adenovirus Late genes + geneIX + Viral Origin + the bacterial ori-amp sequence of pBR322 Ppack10Adenovirus Late genes + Viral Origin + the bacterial ori-amp sequence ofpBR322 Ppack11 Subset of Adenovirus Late genes + gene IX + ViralOrigin + the bacterial ori-amp sequence of pBR322 Ppack12 Subset ofAdenovirus Late genes + Viral Origin + the bacterial ori-amp sequence ofpBR322

Adenoviral Packing Cell (PC)

A PC of the present disclosure is not designed to replicate wild typeAdenovirus.

In an embodiment, one isolated nucleic acid (NA) is stably integratedinto a HC to create a PC expressing Adenoviral early region genes (seeTable 3—PC1, PC2, PC3 and PC4). In an embodiment, two isolated nucleicacids (NAs) are stably integrated into a HC to create a PC expressingAdenoviral early region genes (see Table 3—PC5, PC6, PC7, PC8, PC9,PC10, PC11, PC12, PC13, PC14, PC15, PC16, PC17, PC18, PC19 and PC20). Insome embodiments, viral early region genes of the PC can be a geneequivalent to an Adenoviral early region gene, for example viral genesthat are functionally equivalent to an Adenoviral early region gene.Examples of viral early genes include, but are not limited to, simian 40(SV40) large tumour antigen (Tag), human cytomegalovirus (HCMV)immediate-early (IE) region 2, and herpes simplex virus. In anembodiment, Adenoviral late genes can be cloned downstream of aninducible promoter. For example, the inducible promoter can be comprisedof a plurality of sequential copies of the tet operator (TetO) sequence.In an embodiment, the inducible promoter comprises seven sequentialcopies of the TetO sequence. In an embodiment, Adenoviral late genes areregulated by a repressed promoter. In an embodiment, a singletransfection system of the present disclosure utilizetetracycline-repressed promoters and repressors originally found on theTn10 transposon in E. coli, in order to silence expression of the Adgenes when the specific transcriptional inducer is not present.

Table 3 lists coding sequences stably integrated into a host cell (HC)to create PCs of the present disclosure. A method for producing anAdenovirus packaging cell line permissive for replication of afully-deleted Adenoviral vector independent of helper Adenovirusincludes introducing into a cell line permissive for Adenovirusreplication an isolated nucleic acid molecule comprising an Adenovirusearly region 1 (E1) coding sequence and an Adenovirus pIX codingsequence, wherein the Adenovirus early region 1 (E1) coding sequence andthe Adenovirus pIX coding sequence are stably integrated into the cellline.

TABLE 3 PC PC for Co-Transfection System - One Isolated NA - FIG. 5 PC1Adenovirus E1 coding sequence PC2 Adenovirus E1 + protein IX codingsequence PC3 Adenovirus E1, Viral Early gene coding sequence PC4Adenovirus E1 + protein IX coding sequence, Viral Early gene PC forSingle Transfection System where Late Genes Expressed under InduciblePromoter - Two Isolated NAs - FIG. 6 PC5 Adenovirus E1 Adenovirus Lategenes + gene IX + Inducible promoter PC6 Adenovirus E1 + Adenovirus Lategenes + Inducible promoter gene IX PC7 Adenovirus E1 Subset ofAdenovirus Late genes + gene IX + Inducible promoter PC8 Adenovirus E1 +Subset of Adenovirus Late genes + Inducible gene IX promoter PC9Adenovirus E1 Adenovirus Late genes + gene IX + Inducible promoter +Inducible viral Early gene + Viral origin of replication PC10 AdenovirusE1 + Adenovirus Late genes + Inducible promoter + gene IX Inducibleviral Early gene + Viral origin of replication PC11 Adenovirus E1 Subsetof Adenovirus Late genes + gene IX + Inducible promoter + Inducibleviral Early gene + Viral origin of replication PC12 Adenovirus E1 +Subset of Adenovirus Late genes + Inducible gene IX promoter + Inducibleviral Early gene + Viral origin of replication PC for SingleTransfection System where Late Genes Regulated by Repressed Promoter -Two Isolated NAs - FIG. 6 PC13 Adenovirus E1, Adenovirus Late genes +gene IX + promoter + Repressor gene tetO PC14 Adenovirus E1 + AdenovirusLate genes + promoter + tetO gene IX, Repres- sor gene PC15 AdenovirusE1, Subset of Adenovirus Late genes + gene IX + Repressor genepromoter + tetO PC16 Adenovirus E1 + Subset of Adenovirus Late genes +promoter + gene IX, Repres- tetO sor gene PC17 Adenovirus E1, AdenovirusLate genes + gene IX + promoter + Repressor gene tetO + Inducible ViralEarly gene + Viral Ori PC18 Adenovirus E1 + Adenovirus Late genes +promoter + tetO + gene IX, Repres- Inducible Viral Early gene + ViralOri sor gene PC19 Adenovirus E1, Subset of Adenovirus Late genes + geneIX + Repressor gene promoter + tetO + Inducible Viral Early gene + ViralOri PC20 Adenovirus E1 + Subset of Adenovirus Late genes + promoter +gene IX, Repres- tetO + Inducible Viral Early gene + Viral Ori sor gene

In an embodiment, the repressor gene is tet Rep, however any repressorand its binding site (“operator”) can be used to generalized.

At least one construct (e.g., pPack, FDV) is transfected into a hostcell that lacks overlapping sequences with the nucleic acids of the hostcell (and with each other if both a pPack and a FDV are co-transfected),the overlapping sequences otherwise enabling homologous recombinationleading to replication competent wild type Adenovirus in the host cellinto which the pPack and the FDV are to be transferred. In anembodiment, an isolated nucleic acid is introduced into a HC for stableintegration of E1A and/or E1B coding sequences into the HC. In anembodiment, an isolated nucleic acid is introduced into a HC for stableintegration of E1A and/or E1B and gene IX coding sequences into the HC.The presence of the gene IX coding sequence encodes for the secondintermediate protein, pIX, which helps stabilize the viral capsid ofGDVs propagated from the PC.

The regulation of the nucleic acid(s) expressed in the PC can beaccomplished at the level of gene structure (e.g., activation by asite-specific recombinase), transcription (e.g., inducible orinhibitable promoter), mRNA stability, or translation. The regulation ofthe nucleic acid(s) expressed in the PC can be by the same or differentsystems. In an embodiment, the viral early gene(s) are silent untilexposure to a transcriptional inducer. Expression of the viral earlygene(s) can then induce expression of Adenoviral late genes. In anembodiment, the expression of at least one viral early gene can turn onthe one or more Adenoviral late genes. In an embodiment, the at leastone viral early gene product can be regulated by a constitutive promoterand the one or more Adenoviral late genes can be regulated directly. Inan embodiment, the at least one viral early gene and the one or moreAdenoviral late genes can be regulated by the same system. In anembodiment, the at least one viral early gene and the one or moreAdenoviral late genes can be regulated by different systems.

In some embodiments, regulation of the at least one viral early genesand the one or more viral late genes is accomplished usingtranscriptional induction, however other methods of regulating genes arepossible using the systems disclosed herein. For example, regulation ofgene expression for the at least one viral early gene and the one ormore viral late genes can be accomplished by one of 1) alteration ofgene structure: site-specific recombinases can activate gene expressionby removing inserted sequences between the promoter and the gene; 2)changes in transcription: either by induction (covered) or by relief ofinhibition; 3) changes in mRNA stability, by specific sequencesincorporated in the mRNA or by siRNA; 4) changes in translation, bysequences in the mRNA; or a combination thereof.

It should be understood that the Adenovirus late genes can be from anyAdenovirus serotype and with any naturally occurring or artificiallyselected tropism. Adenoviral serotypes differ in their natural tropismand immunogenicity. The various serotypes of Adenovirus have been foundto differ in at least their capsid proteins (e.g., penton-base and hexonproteins), proteins responsible for cell binding (e.g., fiber proteins),and proteins involved in Adenovirus replication. These differences intropism and capsid proteins among serotypes have led to the manyresearch efforts aimed at redirecting the Adenovirus tropism bymodification of the fiber proteins and immune responses by modificationof hexon and penton proteins. It has been found that the Adenovirus lategenes products code for the penton-base, hexon proteins and the fiberproteins. In an embodiment, natural variations in the late genes thatencode for the penton-base, hexon proteins and/or the fiber proteins,affect the serotype (immune response) and/or tropism. In an embodiment,engineered variations in the late genes that encode for the penton-base,hexon proteins and/or the fiber proteins, affect the serotype and/ortropism.

A change in serotype can mitigate immune reactions. The epitopes locatedon the hexon protein of the Adenovirus provide the basis for theclassification of Adenoviruses into the 51 serotypes known to date(Journal of Virology, October 2005; 79(20): 12635-12642). In anembodiment, the Adenovirus late genes include naturally occurring hexongene variants, thus affecting the Adenovirus serotype. In an embodiment,the Adenovirus late genes are chimeric, where the hexon gene of oneAdenovirus serotype are replaced with the hexon gene of anotherAdenovirus serotype. Such chimeric Adenovirus vectors have beenengineered, for example, by replacing the Ad5 hexon gene with the hexongene of A3 to create an Ad5/H3 vector (Journal of Virology, December2002; 76(24): 12775-12782).

In an embodiment, the Adenovirus late genes include modifications to thepenton base and/or fiber protein, altering the tropism of theAdenovirus. For example, Ad5 vectors having capsid mutations orpseudotyped with the short fiber from serotype 41 (Ad41s) have beenfound to mediate very low liver transduction (Molecular Therapy, August2004; 10(2): 344-354). Similarly, it has been shown that when chimericfiber proteins in which the head domains of Ad5 and Ad3 are exchanged,the chimeric fiber containing the Ad5 fiber head domain blocked thebinding of Ad5 fiber but not Ad3 fiber, and the chimeric fibercontaining the Ad3 fiber head blocked the binding of labeled Ad3 fiberbut not Ad5 fiber (Journal of Virology, May 1995; 69(5): 2850-2857).Also, when an Adenovirus vector containing chimeric fibers composed ofthe tail and shaft domains of Adenovirus serotype 5 and the knob domainof serotype 3 were generated, the receptor recognition profile of thevirus containing the fiber chimera was altered (Journal of Virology,October 1996; 70(10): 6839-6846).

In an embodiment, the Adenovirus late genes include selected mutationsto the penton base and/or fiber protein, altering the tropism of theAdenovirus. For example, Ad5 vectors having capsid mutations orpseudotyped with the short fiber from serotype 41 (Ad41s) have beenfound to mediate very low transduction to liver cells (MolecularTherapy, August 2004; 10(2): 344-354). A fiber mutant, F/K20, that has alinker and a stretch of 20 lysine residues added at the C terminus ofthe fiber, showed a remarkably enhanced efficiency in genetictransduction of human glioma cells (Human Gene Therapy, 1998; 9(17):2503-251). Epithelial and endothelial cells expressing the primaryCoxsackie virus B Adenovirus (Ad) receptor (CAR) and integrincoreceptors are natural targets of human Ad infections. The fiber knobof Adenoviral A, C, D, E and F Ad serotypes binds CAR by mimicking theCAR-homodimer interface, and the penton base containingarginine-glycine-aspartate (RGD) motifs binds with low affinity to αvintegrins inducing cell activation. Researchers generated sevendifferent genetically modified Ad vectors with RGD sequences insertedinto the HI loop of fiber knob. All mutants bound and infected CAR andαv integrin-positive epithelial cells with equal efficiencies. However,the mutant Adenoviruses containing two additional cysteines, both N andC terminals of the RGD sequence (RGD-4C), were uniquely capable oftransducing CAR-less hematopoietic and nonhematopoietic human tumor celllines and primary melanoma cells (Gene Therapy, 2003; (10): 1643-1653).

In spite of its broad host range, Adenovirus type 5 (Ad5) transduces anumber of clinically relevant tissues and cell types inefficiently,mostly because of low expression of the coxsackievirus-Adenovirusreceptor (CAR). To improve gene transfer to such cells, researchersmodified the Ad5 fiber knob to recognize novel receptors. Theresearchers expressed a functional Ad5 fiber knob domain on the capsidof phage and employed this display system to construct a largecollection of ligands in the HI loop of the Ad5 knob. Panning thislibrary on the CAR-negative mouse fibroblast cell line NIH 3T3 resultedin the identification of three clones with increased binding to thesecells. Adenoviruses incorporating these ligands in the fiber genetransduced NIH 3T3 cells 2 or 3 orders of magnitude better than theparent vector. The same nonnative tropism was revealed in other celltypes, independently of CAR expression. These Ad5 derivatives provedcapable of transducing mouse and human primary immature dendritic cellswith up to 100-fold increased efficiency. (Journal of Virology, October2003; 77(20): 11094-11104).

In an embodiment, the Adenovirus late genes include peptide or molecularadaptors that target Adenoviral late genes to selective tissue“addresses”, altering the tropism of the Adenovirus. For example,researchers linked the Fab fragments of monoclonal antibodies that bindto Adenovirus type 5 (Ad5) to a synthetic lung-homing peptide (GFE-1peptide) and tested the ability of the resulting bispecific conjugate toretarget Ad5. Cells that express the receptor for the GFE-1 peptide andare resistant to Ad5 infection were sensitized to recombinant Ad5vectors in the presence of the Fab-GFE adaptor (Human Gene Therapy, Sep.20, 2000: 11(14): 1971-1981). Other researchers have designed ligandscorresponding to prototypes of the most represented families ofphagotopes recovered from intracellular phages, and individuallyinserted these ligands into Ad5-green fluorescent protein (GFP) (AdGFP)vectors at the extremities of short fiber shafts (seven repeats [R7])terminated by scissile knobs. Results validated the concept ofdetargeting and retargeting Ad vectors via a deknobbing system andredirecting Ad vectors to an alternative endocytic pathway via a peptideligand inserted in the fiber shaft domain (Journal of Virology, July2004; 78(13): 7227-7247). Still others have tested the efficiency ofhuman Adenovirus serotype 5 (Ad5) transgene delivery on several humanand animal cell lines in vitro, by using a bimodular 35-mer oligopeptidecarrying two peptide domains with different ligand specificities. Onedomain mimicked the fiber knob-binding region of the alpha₂ domain ofhuman MHC1 molecules (MH20), and the other corresponded to thegastrin-releasing peptide (GRP). Two synthetic peptides with differentconfigurations were analyzed in Ad-mediated gene transfer assays usingAd5Luc3 vector carrying the luciferase reporter gene. One peptide(GRP-MH2O) had the GRP domain on the N-terminal side of MH20, while theother (MH20-GRP), the C-terminally amidified GRP, was on the C-terminalside of MH20. The GRP-MH20 peptide, but not MH20-GRP, was capable ofenhancing luciferase gene delivery to Ad-susceptible cells in a GRPreceptor-dependent manner More importantly, GRP-MH20 could also confersusceptibility to Ad infection to normal or cancer cells that lack fiberreceptors for the virus. The data suggested that GRP receptors couldfunction efficiently as alternative attachment receptors for Ad5, butthat Ad5 bound to GRP receptors still depended, at least partially, onthe penton base-mediated endocytotic pathway for subsequent cell entry(Human Gene Therapy, Nov. 10, 1999; 10(16): 2577-2586) and (VirusResearch, March 2001; 73(2): 145-152).

Regulation of Gene Expression

An inducible system is off unless there is the presence of some molecule(called an inducer) that allows for gene expression. The molecule issaid to “induce expression”. The manner in which this happens isdependent on the control mechanisms as well as differences betweenprokaryotic and eukaryotic cells. A repressible system is on except inthe presence of some molecule (called a corepressor) that suppressesgene expression. The molecule is said to “repress expression”. Themanner in which this happens is dependent on the control mechanisms aswell as differences between prokaryotic and eukaryotic cells.

Gene regulation systems/control systems for gene expression useful inpracticing the present invention include, but are not limited to, theTet System, the ecdysone receptor-based RheoSwitch mammalian inducibleexpression system (Ecdysteroid/muristerone/retinoid X receptor),FK1012/FK506-induced system, the Rapamycin/FKBP12/FRAP system,RU486(mifepristone)/GLVP/PR-LBDA system, Cytochrome P-450, Ga14,Streptogramin/macrolide induced system, Acylated homoserine lactone(AHL) induced system and UTR aptamers. In an embodiment, the presentinvention uses the Tet System with the genes encoding for VP16-TetRfusion protein as Transcriptional Activator, TetO as promoter, and atleast one of tetracycline (tc) or doxycycline (dox) as the inducer.

Late Genes Expressed Under Inducible Promoter

A major area of interest concerns the problem that most gene-basedmodification approaches are functionally equivalent to digital switches:the genes exist either in a wild-type or in a modified state, withlittle possibility of a partial modification or of reversibility. Toaddress this, many studies have investigated inducible-promoter systems.These systems use a regulating agent (including small molecules such astetracycline, peptide and steroid hormones, nerotransmitters, andenvironmental factors such as heat, and osmolarity) to induce or tosilence a gene. Such systems are analog in the sense that the responseis graduated, being dependent on the concentration of the regulatingagent. Also, such systems are reversible with the withdrawal of theregulating agent. While a number of approaches have been investigated,including cold-inducible gene regulation systems, and single regulatedpromoters (such as those regulated by heavy metals, heat shock, orsteroids), finer regulatory control has been achieved by binary systemscomposed of an effector molecule (the regulator) and a target transgene.A prototypic system is the “tetracycline-responsive system” or “Tet-OFFsystem” originally developed by Gossen and Bujard (Proc. Natl. Acad.Sci. USA 1992; 89: 5547-5551). The Tet-OFF system uses an effectorprotein composed of a fusion between the Escherichia colitetracycline-repressor protein (TetR) and the Herpes simplex VP16 transactivation domain (VP16-TetR fusion protein). In the absence oftetracycline (tc) or doxycycline (dox), the fusion protein binds the19-bp operator sequence, TetO. In the presence of tc or dox, therepressor dissociates from TetO and activation is lost.

Late Genes Regulated by Repressed Promoter

Another system referred to as the “reverse tetracycline” or the “Tet-ON”system, works by activating transcription when tc, dox oranhydrotetracycline is present and was developed by Gossen et al.(Science, 1995; 268: 1766-1769). The Tet-On system uses a mutated TetRsuch that, for example, binding of dox or tc induces DNA binding ratherthan abrogating it, rTetR. VP16-rTetR fusion is then an activated onlyin the presence of tc, dox or anhydro-tc. The packaging systemsdisclosed herein use the “Tet-ON” system.

FIG. 5 is a diagrammatic representation showing an embodiment of aco-transfection system comprising a fully-deleted Adenoviral vectorconstruct (FDV), a packing construct (pPack), and an Adenoviruspackaging cell (PC) for producing encapsidated encapsidatedfully-deleted Adenovirus-based gene transfer vectors (GDVs) withouthelper Adenovirus. FIG. 6 is a diagrammatic representation showing anembodiment of a transfection system comprising a fully-deletedAdenoviral vector construct (FDV) and an Adenovirus packaging cell (PC)for producing encapsidated fully-deleted Adenovirus-based gene transfervectors (GDVs) without helper Adenovirus. Table 4 lists variousembodiments of a system (including a PC and at least one construct (aFDV or a pPack) to propagate GDVs of the present disclosure. At leasttwenty-eight (28) embodiments of systems for propagating GDVs of thepresent invention are contemplated, as summarized in Table 4 below. Amethod for propagating an adenoviral vector includes (a) providing anAdenovirus packaging cell line; (b) transfecting a fully-deletedAdenoviral vector construct into the cell line; and optionally (c)transfecting a packaging construct into the cell line, wherein thefully-deleted Adenoviral vector construct and optionally the packagingconstruct can transfect the Adenovirus packaging cell line resulting inthe encapsidation of a fully-deleted Adenoviral vector independent ofhelper Adenovirus.

TABLE 4 Embodiments of Systems for Propagating GDVs A system of thepresent invention includes a PC, a FDV, and optionally a pPack. andoptionally the If the PC is then the FDV is pPack is: PC1, FDV1, pPack1.PC2, FDV1, pPack2. PC1, FDV2, pPack3. PC2, FDV2, pPack4. PC1, FDV1,pPack5. PC2, FDV1, pPack6. PC1, FDV2, pPack7. PC2, FDV2, pPack8. PC3,FDV1, pPack9. PC4, FDV1, pPack10. PC3, FDV2, pPack11. PC4, FDV2,pPack12. PC5, FDV3, None. PC6, FDV3, None. PC7, FDV4, None. PC8, FDV4,None. PC9, FDV3, None. PC10, FDV3, None. PC11, FDV4, None. PC12, FDV4,None. PC13, FDV1, None. PC14, FDV1, None. PC15, FDV1, None. PC16, FDV1,None. PC17, FDV3, None. PC18, FDV3, None. PC19, FDV3, None. PC20, FDV3,None.

A GDV propagated by a system of the present disclosure includes bothAdenoviral inverted terminal repeats (LITR and RITR) separated byapproximately 28 to 37 kb, the viral packaging signal (Ψ), and at leastone DNA insert (all or a fragment of at least one gene of interest(GOI)) which comprises a gene sequence encoding a protein of interest.No viral structural genes are contained in the GDV. In an embodiment, aGOI can be a therapeutic gene, immunomodulatory gene, a vaccine gene ora combination thereof. In an embodiment, a GDV of the present disclosureexpresses an immunomodulatory gene. In an embodiment, theimmunomodulatory gene is a gene encoding human CD8. In an embodiment, aGDV of the present disclosure expresses a therapeutic gene. In anembodiment, the therapeutic gene is an interleukin. In an embodiment,the therapeutic gene is factor VIII. In some embodiments of the presentdisclosure, a GDV comprises one or more transgenes encoding therapeuticmolecules of interest along with a CD8 polypeptide described herein.

Administration of GDVs

One skilled in the art will appreciate that suitable methods ofadministering a GDV of the present disclosure to an animal fortherapeutic purposes, e.g., gene therapy, immunosuppressive therapy,vaccination, and the like (see, for example, Rosenfeld et al., Science,252, 431 434 (1991), Jaffe et al., Clin. Res., 39(2), 302A (1991),Rosenfeld et al., Clin. Res., 39(2), 311A (1991), Berkner,BioTechniques, 6, 616 629 (1988)), are available, and, although morethan one route can be used to administer the GDV, a particular route canprovide a more immediate and more effective reaction than another route.Pharmaceutically acceptable excipients are also well-known to those whoare skilled in the art, and are readily available. The choice ofexcipient will be determined in part by the particular method used toadminister the GDV. Accordingly, there is a wide variety of suitableformulations of the GDVs of the present invention. The followingformulations and methods are merely exemplary and are in no waylimiting. However, oral, injectable and aerosol formulations arepreferred.

Formulations suitable for oral administration can consist of (a) liquidsolutions; (b) capsules, sachets or tablets; (c) suspensions in anappropriate liquid; and (d) suitable emulsions. In an embodiment, theGDVs of the present invention, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. The GDVs ofthe present invention may also be formulated as pharmaceuticals fornon-pressured preparations such as in a nebulizer or an atomizerFormulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can compriseanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The dose administered to an animal, particularly a human, in the contextof the present invention will vary with the gene or other sequence ofinterest, the composition employed, the method of administration, andthe particular site and organism being treated. The dose should besufficient to effect a desirable response, e.g., therapeutic or immuneresponse, within a desirable time frame.

Hence, one or more of the following routes may administer the GDVs ofthe present disclosure: oral administration, injection (such as directinjection), topical, inhalation, parenteral administration, mucosaladministration, intramuscular administration, intravenousadministration, subcutaneous administration, intraocular administrationor transdermal administration. In an embodiment, encapsidated FDVs ofthe present disclosure are administered via injection. In an embodiment,GDVs of the present disclosure are administered topically. In anembodiment, GDVs of the present disclosure are administered byinhalation. In an embodiment, GDVs of the present disclosure areadministered by one or more of: parenteral, mucosal, intramuscular,intravenous, subcutaneous, intraocular or transdermal administrationmeans, and are formulated for such administration.

Typically, a physician will determine the actual dosage of GDVs thatwill be most suitable for an individual subject and it will vary withthe age, weight and response of the particular patient and severity ofthe condition. The specific dose level and frequency of dosage for anyparticular patient may be varied and will depend upon a variety offactors including the activity of the specific compound employed, themetabolic stability and length of action of that compound, the age, bodyweight, general health, sex, diet, mode and time of administration, rateof excretion, drug combination, the severity of the particularcondition, and the host undergoing therapy.

The dose administered to an animal, particularly a human, in the contextof the present invention will vary with the therapeutic transgene ofinterest and/or the nature of the immunomodulatory molecule, thecomposition employed, the method of administration, and the particularsite and organism being treated. However, preferably, a dosecorresponding to an effective amount of a GDV is employed. An “effectiveamount” is one that is sufficient to produce the desired effect in ahost, which can be monitored using several end-points known to thoseskilled in the art. For instance, one desired effect is nucleic acidtransfer to a host cell. Such transfer can be monitored by a variety ofmeans, including, but not limited to, a therapeutic effect (e.g.,alleviation of some symptom associated with the disease, condition,disorder or syndrome being treated), or by evidence of the transferredgene or coding sequence or its expression within the host (e.g., usingthe polymerase chain reaction, Northern or Southern hybridizations, ortranscription assays to detect the nucleic acid in host cells, or usingimmunoblot analysis, antibody-mediated detection, or particularizedassays to detect protein or polypeptide encoded by the transferrednucleic acid, or impacted in level or function due to such transfer).These methods described are by no means all-inclusive, and furthermethods to suit the specific application will be apparent to theordinary skilled artisan. In this regard, it should be noted that theresponse of a host to the introduction of a GDV can vary depending onthe dose of virus administered, the site of delivery, and the geneticmakeup of the GDV as well as the transgene and the means of inhibitingan immune response.

Generally, to ensure effective transfer of the GDVs of the presentinvention, it is preferable that about 1 to about 5,000 copies of theGDV according to the invention be employed per cell to be contacted,based on an approximate number of cells to be contacted in view of thegiven route of administration, and it is even more preferable that about3 to about 300 pfu enter each cell. However, this is merely a generalguideline, which by no means precludes use of a higher or lower amount,as might be warranted in a particular application, either in vitro or invivo. Similarly, the amount of a means of inhibiting an immune response,if in the form of a composition comprising a protein, should besufficient to inhibit an immune response to the recombinant GDVcomprising the transgene. For example, the actual dose and schedule canvary depending on whether the composition is administered in combinationwith other pharmaceutical compositions, or depending on interindividualdifferences in pharmacokinetics, drug disposition, and metabolism.Similarly, amounts can vary in in vitro applications, depending on theparticular cell type targeted or the means by which the GDV istransferred. One skilled in the art easily can make any necessaryadjustments in accordance with the necessities of the particularsituation.

Immunosuppressive Therapy for Allogeneic Transplantation

A major unmet need in transplant immunobiology is the development ofmore specific immune inhibition strategies directed exclusively to thealloantigen response. Ideally, such strategies would inhibitallorejection without the need for continuous general pharmacologicimmunosuppression and its attendant complications and costs. Achievingspecific immunological tolerance to such alloantigens can improvetransplant longevity and quality of life for the recipient, and at thesame time considerably improve the cost effectiveness of transplanttherapy. The present invention achieves these heretofore elusive goals.

The veto effect mediated by targeted expression of CD8α can effectivelyand specifically suppress responding CD4⁺ T cells (MHC classII-restricted) as well as CD8⁺ T cells (MHC class I-restricted),therefore both the cellular and humoral components of the immuneresponse directed against alloantigens can be inhibited. Adenoviralvectors are particularly suited for transduction of the CD8 gene toallogeneic cells/tissues for transplantation because they infect a widerange of cell and tissues with high efficiency and because thetransduced DNA is expressed transiently and not permanently integratedinto the genome of the transduced cells. For CD8 mediated veto immuneprotection of allogeneic transplants based on Adenoviral vectortransduction of CD8 to the allogeneic transplant, it is desirable thatthe Adenoviral vector carry no Adenoviral genes, as the consequencewould be long-term immune non-responsiveness to the base Adenovirus.

In an embodiment, the GDVs of the present disclosure are engineered totransform cells and tissues into specifically immune suppressivemoieties. In an embodiment, the cells are human keratinocytes and a skintransplant can be engineered to treat, for example, a burn victim or anindividual with a diabetic ulcer. In an embodiment, the cells arenonhuman primate hepatocytes and a hepatocyte transplant can beengineered to treat, for example, end-stage liver disease or a genedefect. In an embodiment, the cells are kidney cells, for example fromporcine or a nonhuman primate, and a kidney transplant can be engineeredto treat, for example, end-stage kidney disease. A GDV vetoes rejectionby inducing suicide (apoptosis) specifically of lymphocytes with theability to reject transplants. In an embodiment, the GDVs of the presentdisclosure find use in immunosuppressive therapy for allogeneictransplants, wherein the transplant is selected from one of pancreaticislets, hepatocytes or keratinocytes.

In an embodiment, methods and compositions are provided for specificallyinhibiting an alloantigen response to donor and/or host antigens,depending on the nature of the allograft, in order to prolong thesurvival of allogeneic grafts and protect the health of the transplantrecipient. Thus, using the compositions and methods described herein onemay selectively inhibit allorejection activity directed to either donoror host antigens without the need for chronic administration of generalimmunosuppressive agents, effectively resulting in specificimmunological tolerance to donor tissue, organs and/or cells.

In one aspect, methods for specifically inhibiting immune responses toalloantigens are provided, comprising contacting a target cellexpressing at least one such alloantigen with a GDV encoding all or afunctional portion of a CD8 polypeptide, preferably a human CD8polypeptide, still more preferably the human CD8 α-chain, whereby theCD8 polypeptide is expressed by the target cell and whereby thealloimmune response directed against the alloantigen is specificallyinhibited. In one embodiment, the alloantigen comprises a donoralloantigen and the target cell comprises an allograft cell. In analternative embodiment, the alloantigen comprises a recipientalloantigen and the target cell comprises a recipient cell. In a furtherembodiment, the alloimmune response includes both a humoral componentand a cellular component. In a preferred embodiment, the alloimmuneresponse is effectively inhibited without the need for generalimmunosuppressive agents. By “nucleic acid molecules encoding CD8”, andgrammatical equivalents thereof is meant the nucleotide sequence ofhuman CD8 as well as nucleotide sequences having at least about 80%sequence identity, usually at least about 85% sequence identity,preferably at least about 90% sequence identity, more preferably atleast about 95% sequence identity and most preferably at least about 98%sequence identity.

In another aspect, methods for specifically inhibiting immune responsesto donor alloantigen are provided, comprising conditioning donorallograft cells in vivo or ex vivo to express all or a functionalportion of a CD8 polypeptide, preferably a human CD8 polypeptide, stillmore preferably the human CD8 α-chain. In one embodiment, theconditioning step comprises contacting the allograft cells in vivo or exvivo with a GDV encoding all or a functional portion of a CD8polypeptide, whereby the CD8 polypeptide is expressed by allograft cellsand whereby the recipient immune response directed against donoralloantigen is specifically inhibited. Preferably, both the cellular andhumoral components of the recipient alloimmune response are effectivelyand specifically inhibited without the need for generalimmunosuppressive agents.

In another aspect, methods for specifically inhibiting immune responsesto recipient alloantigen are provided, comprising in vivo conditioningof recipient cells to express all or a functional portion of a CD8polypeptide, preferably a human CD8 polypeptide, still more preferablythe human CD8α-chain. Preferred recipient cells for the subjectconditioning step include those found in the recipient tissues andorgans most at risk of a GVHD immune response such as, e.g., liver, skinand intestinal tract. In one embodiment, the conditioning step comprisescontacting such recipient cells in vivo with a GDV encoding all or afunctional portion of a CD8 polypeptide, whereby the CD8 polypeptide isexpressed by the cells and whereby the donor immune response directedagainst recipient alloantigen is specifically inhibited. Preferably, thedonor alloimmune response is effectively and specifically inhibitedwithout the need for general immunosuppressive agents.

Also provided are methods for prolonging the survival of an allograft ina recipient, comprising conditioning the allograft cells in vivo or exvivo to express all or a functional portion of a CD8 polypeptide,preferably a human CD8 polypeptide, still more preferably the humanCD8α-chain. In one embodiment, the conditioning step comprisescontacting the allograft cells in vivo or ex vivo with a GDV encodingall or a functional portion of a CD8 polypeptide, wherein the CD8polypeptide is expressed by allograft cells and whereby the survivaltime of the allograft in the recipient is extended. Preferably, theconditioning step is performed prior to or contemporaneously withtransplantation of the allograft. Still more preferably, theconditioning step is performed ex vivo prior to transplantation of theallograft, or in vivo in the donor prior to or contemporaneous withharvesting of the allograft. Most preferably, use of the subject methodsis effective to induce stable immunological tolerance to the allograft,such that chronic administration of general immunosuppressive agentswill not be required.

Also provided are methods for suppressing GVHD in a recipient,comprising in vivo conditioning of recipient cells at risk of a GVHDimmune response to express all or a functional portion of a CD8polypeptide, preferably a human CD8 polypeptide, still more preferablythe human CD8α-chain. In one embodiment, the conditioning step comprisescontacting recipient cells in vivo with a GDV encoding all or afunctional portion of a CD8 polypeptide, whereby the CD8 polypeptide isexpressed by the cells and whereby the GVHD immune response raisedagainst the recipient cells by transplanted donor T cells is suppressed.Preferably, the conditioning step is performed contemporaneously with orsubsequent to transplantation of the allograft. Still more preferably,the conditioning step is performed in vivo in the recipient aftertransplantation of the allograft. Most preferably, use of the subjectmethods is effective to induce stable immunological tolerance oftransplanted donor T cells to recipient alloantigen, such that chronicadministration of general immunosuppressive agents is not needed.

Preferred CD8 polypeptides for use in the subject methods andcompositions will generally comprise the CD8α-chain, more preferably theextracellular domain of the CD8α-chain, and still more preferably theIg-like domain of the CD8α-chain. In alternative preferred embodiments,the CD8 polypeptides may comprise or consist essentially of theextracellular domain of the CD8α-chain and a transmembrane domain, ormore preferably the Ig-like domain of the CD8α-chain and a transmembranedomain. In a particularly preferred embodiment, the transmembrane domainis the transmembrane domain of the CD8α-chain. Given the nature of thesubject expression methods, as well as the apparent inadequacies of theprior art soluble forms of CD8α-chain described above, the presence ofthe CD8α-chain transmembrane domain or a suitable alternativetransmembrane region is deemed essential.

In a further aspect, the present invention provides improved transplantallografts capable of specifically and effectively inhibiting arecipient immune response raised against them. In one embodiment, theimproved transplant allograft comprises allograft cells modified toexpress a CD8 polypeptide, preferably a human CD8 polypeptide, stillmore preferably the human CD8α-chain. As discussed herein, the CD8polypeptide may comprise or alternatively consist essentially of theextracellular domain of the CD8α-chain and a transmembrane domain, orthe Ig-like domain of the CD8α-chain and a transmembrane domain. Thetransmembrane domain may be that of the CD8α-chain or may be anotheradvantageously-selected transmembrane domain. Modification of allograftcells may be achieved with a liposome-mediated nucleic acid transfervehicle, a viral-mediated nucleic acid transfer vehicle, and the like,as disclosed herein.

In a still further aspect, an improved organ preservation solution isprovided, comprising a GDV encoding a CD8 polypeptide. Thus, in apreferred embodiment, the invention provides an improved organpreservation solution comprising a GDV comprising a nucleic acidencoding for a CD8 polypeptide, preferably a human CD8 polypeptide, andmost preferably the human CD8α-chain. In another preferred embodiment,the improved organ preservation solution comprises a GDV comprising anucleic acid encoding for the extracellular domain of a CD8α-chain and atransmembrane domain, or alternatively the Ig-like domain of theCD8α-chain and a transmembrane domain. In a particularly preferredembodiment, the transmembrane domain is the CD8α-chain transmembranedomain. In further embodiments, the GDV may further comprise a nucleicacid encoding for an anti-inflammatory molecule such as, e.g., hemeoxygenase.

In a broader aspect, methods are provided for specifically inhibiting ahost immune response to a target cell-specific antigen, comprisingconditioning the target cell in vivo or ex vivo to express all or afunctional portion of a CD8 polypeptide, more preferably the human CD8polypeptide, still more preferably the human CD8α-chain, wherein the CD8polypeptide is expressed by the target cell and whereby an immuneresponse directed against such antigen is specifically inhibited. In oneembodiment, the target cell-specific antigen is an alloantigen. Inanother embodiment, the target cell-specific antigen is an autoantigen.In a preferred embodiment, the conditioning step comprises contactingthe target cell in vivo or ex vivo with a GDV encoding the CD8polypeptide.

In a still further aspect, methods for preventing the development of andfor treating autoimmune diseases are provided, comprising administeringto a patient in need thereof a therapeutic composition comprising a GDVencoding all or a functional portion of a CD8 polypeptide, preferably ahuman CD8 polypeptide, still more preferably the CD8α-chain, whereinexpression of the CD8 polypeptide by a contacted target cellspecifically inhibits an autoreactive immune response directed againstthe target-cell specific autoantigens.

In order to effect expression of the immunomodulatory molecule (e.g.CD8α-chain) and/or additional therapeutic proteins, the GDVs must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. Once the GDV hasbeen delivered into the cell the nucleic acid encoding the desiredoligonucleotide or polynucleotide sequences may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the construct may be stably integrated into the genome of thecell. This integration may be in the specific location and orientationvia homologous recombination (gene replacement) or it may be integratedin a random, non-specific location (gene augmentation). In further andpreferred embodiments, the nucleic acid may be stably maintained in thecell as a separate, episomal segment of DNA. Such nucleic acid segmentsor “episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

Although the above examples describe a GDV encoding an immunomodulatorygene encoding all or a functional portion of a CD8 polypeptide, itshould be understood that additional immunomodulatory genes encodingimmunomodulatory molecules are contemplated, including, but not limitedto, IL-10, TGF-beta, IL-2, IL-12, IL-15, IL-18, IL-4 and GM-CSF.

Gene Therapy for Gene and Protein Expression

Gene therapy generally involves the introduction into cells oftherapeutic genes, also known as transgenes, whose expression results inamelioration or treatment of genetic disorders. The therapeutic genesinvolved may be those that encode proteins, structural or enzymaticRNAs, inhibitory products such as antisense RNA or DNA, or any othergene product. Expression is the generation of such a gene product or theresultant effects of the generation of such a gene product. Thus,enhanced expression includes the greater production of any therapeuticgene or the augmentation of that product's role in determining thecondition of the cell, tissue, organ or organism.

In general, the instant invention relates to GDVs for transferringselected genetic material of interest (e.g., DNA or RNA) to cells invivo. The invention also relates to methods of gene therapy using thedisclosed GDVs and genetically engineered cells produced by the method.Diseases that may be treated by the present invention include, but arenot limited to, Hemophilia A (with Factor VIII), Parkinson's Disease,Congestive Heart Failure and Cystic Fibrosis. In an embodiment, a GDV ofthe present disclosure carrying at least a fragment of a gene ofinterest can infect the myocardium in vivo following intracardiacinjection. In an embodiment, a GDV of the present disclosure carrying atleast a fragment of the CFTR gene can be introduced in situ into thelungs of a Cystic Fibrosis patient by aerosol inhalation. In anembodiment, a GDV of the present disclosure carrying at least a fragmentof a gene coding for Factor VIII can be introduced in situ into a musclein the arm of a patient with Hemophilia A. In an embodiment, a GDV ofthe present disclosure carrying at least a fragment of the ADA gene canbe transduced into a subpopulation of bone marrow cells ex vivo, andthen the transduced bone marrow cells can be transplanted into a patientsuffering from Adenosine deaminase (ADA) deficiency.

The particular therapeutic gene encoded by a GDV of the presentdisclosure is not limiting and includes those useful for varioustherapeutic and research purposes, as well as reporter genes andreporter gene systems and constructs useful in tracking the expressionof transgenes and the effectiveness of Adenoviral and Adenoviral vectortransduction. Thus, by way of example, the following are classes ofpossible genes whose expression may be enhanced by using a GDV of thepresent disclosure: developmental genes (e.g. adhesion molecules, cyclinkinase inhibitors, Wnt family members, Pax family members, Winged helixfamily members, Hox family members, cytokines/lymphokines and theirreceptors, growth or differentiation factors and their receptors,neurotransmitters and their receptors), oncogenes (e.g. ABLI, BLC1,BCL6, CBFA1, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOX,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN,NRAS, PIM1, PML, RET, SRC, TAL1, TCL3 and YES), tumor suppresser genes(e.g. APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53 and WT1),enzymes (e.g. ACP desaturases and hycroxylases, ADP-glucosepyrophorylases, ATPases, alcohol dehycrogenases, amylases,amyloglucosidases, catalases, cellulases, cyclooxygenases,decarboxylases, dextrinases, esterases, DNA and RNA polymerases,hyaluron synthases, galactosidases, glucanases, glucose oxidases,GTPases, helicases, hemicellulases, hyaluronidases, integrases,invertases, isomersases, kinases, lactases, lipases, lipoxygenases,lyases, lysozymes, pectinesterases, peroxidases, phosphatases,phospholipases, phophorylases, polygalacturonases, proteinases andpeptideases, pullanases, recombinases, reverse transcriptases,topoisomerases, xylanases), reporter genes (e.g. Green fluorescentprotein and its many color variants, luciferase, CAT reporter systems,Beta-galactosidase, etc.), blood derivatives, hormones, lymphokines(including interleukins), interferons, TNF, growth factors,neurotransmitters or their precursors or synthetic enzymes, trophicfactors (such as BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, andthe like), apolipoproteins (such as ApoAI, ApoAIV, ApoE, and the like),dystrophin or a minidystrophic, tumor suppressor genes (such as p53, Rb,Rap1A, DCC, k-rev, and the like), genes coding for factors involved incoagulation (such as factors VII, VI, IX, and the like), suicide genes(such as thymidine kinase), cytosine deaminase, or all or part of anatural or artificial immunoglobulin (Fab, ScFv, and the like). Otherexamples of therapeutic genes include fus, interferon α, interferon β,interferon γ, and ADP (Adenoviral death protein).

The therapeutic gene can also be an antisense gene or sequence whoseexpression in the target cell enables the expression of cellular genesor the transcription of cellular mRNA to be controlled. Such sequencecan, for example, be transcribed in the target cell into RNAscomplementary to cellular mRNAs. The therapeutic gene can also be a genecoding for an antigenic peptide capable of generating an immune responsein man. In this particular embodiment, the disclosure makes it possibleto produce vaccines enabling humans to be immunized, in particularagainst microorganisms, viruses and cancer.

Various enzyme genes are also considered therapeutic genes. Particularlyappropriate genes for expression include those genes that are thought tobe expressed at less than normal level in the target cells of thesubject mammal Examples of particularly useful gene products include,but are not limited to, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase, andarginase. Other desirable gene products include fumarylacetoacetatehydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin,glucose-6-phosphatase, low-density-lipoprotein receptor, porphobilinogendeaminase, factor VIII, factor IX, cystathione .beta.-synthase, branchedchain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase,propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoAdehydrogenase, insulin, β-glucosidase, pyruvate carboxylase, hepaticphosphorylase, phosphorylase kinase, glycine decarboxylase (alsoreferred to as P-protein), H-protein, T-protein, Menkes diseasecopper-transporting ATPase, and Wilson's disease copper-transportingATPase. Other examples of gene products include, but are not limited to,cytosine deaminase, hypoxanthine-guanine phosphoribosyltransferase,galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,glucocerbrosidase, sphingomyelinase, α-L-iduronidase,glucose-6-phosphate dehydrogenase, HSV thymidine kinase and humanthymidine kinase. Hormones are another group of genes that may be usedin the Adenoviral-derived vectors described herein. Included are growthhormone, prolactin, placental lactogen, luteinizing hormone,follicle-stimulating hormone, chorionic gonadotropin,thyroid-stimulating hormone, leptin, adrenocorticotropin (ACTH),angiotensin I and II, β-endorphin, β-melanocyte stimulating hormone(β-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitorypeptide (GIP), glucagon, insulin, lipotropins, neurophysins,somatostatin, calcitonin, calcitonin gene related peptide (CGRP),β-calcitonin gene related peptide, hypercalcemia of malignancy factor(1-40), parathyroid hormone-related protein (107-139) (PTH-rP),parathyroid hormone-related protein (107-111) (PTH-rP), glucagon-likepeptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM,secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin(AVP), vasotocin, enkephalinamide, metorphinamide, alpha melanocytestimulating hormone (alpha-MSH), atrial natriuretic factor (5-28) (ANF),amylin, amyloid P component (SAP-1), corticotropin releasing hormone(CRH), growth hormone releasing factor (GHRH), luteinizinghormone-releasing hormone (LHRH), neuropeptide Y, substance K(neurokinin A), substance P and thyrotropin releasing hormone (TRH).Other classes of genes that are contemplated to be inserted into theGDVs of the present disclosure include, but are not limited to,interleukins and cytokines, including interleukin 1 (IL-1), IL-2, IL-3,IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.

Diseases that may be treated by the present invention include, but arenot limited to, prevalent genetic diseases such as Phenylketonuria(phenylalanine-L-monooxygenase), adenosine deaminase deficiency, cysticfibrosis (cystic fibrosis conductance regulator), Parkinson's disease,ornithine caramyltransferase deficiency (OTC), hemophilias (FactorIX-deficiency, Factor VIII-deficiency), Tay-Sachs(N-acetyl-hexosamimidase A), cystic fibrosis, which would involve thereplacement of the cystic fibrosis conductance regulator gene, and otherlipid storage diseases. In addition, the gene encoding erythropoietin(EPO) can used. A FDHIV of the present disclosure is free of Adenoviralearly region and late genes.

A GDV of the present disclosure can be used for treatment ofhyperproliferative disorders such as rheumatoid arthritis or restenosisby transfer of genes encoding angiogenesis inhibitors or cell cycleinhibitors. Transfer of prodrug activators such as the HSV-TK gene canbe also be used in the treatment of hyperploiferative disorders,including cancer. In an embodiment, a GDV of the present disclosureincludes a therapeutic gene sequence and a CD8 gene sequence, whereinthe CD8 gene sequence is capable of preventing an immune response to thetherapeutic gene sequence, as described below. Such applicationsinclude, but are not limited to, Factor VIII deficiency (Hemophilia A)where the patients do not produce any protein from the deficient gene(null alleles). In these patients, an immune response may be mountedagainst the product of the therapeutic gene, just as an immune responsefrequently occurs in Hemophilia A patients treated by injection ofFactor VIII protein.

Adenovirus vectors have been used to produce recombinant proteins usefulin treating diseases. However, contamination of the therapeuticrecombinant proteins with

Adenoviral proteins and/or Adenovirus is a continual problem.Accordingly, there is a need for systems that support growth ofAdenoviral-derived vectors with reduced complement of Ad genes and/orabsence of contamination with replication competent, or helper,Adenovirus. Co-ordinate expression of polypeptides for multimericproteins can be coordinated by co-expression from an Adenoviral vector.For example the expression of equimolar amounts of immunoglobulin heavyand light chains is facilitated by co-ordinate expression from a commonAdenoviral vector.

According to aspects illustrated herein, in an embodiment a proteinexpression protocol includes administering a GDV of the presentdisclosure.

Vaccine Development

In an embodiment, the invention relates to biotechnology and thedevelopment and manufacture of vaccines. The invention is particularlyuseful for the production of vaccines to aid in protection against viraland bacterial pathogens for vertebrates, in particular mammalians andespecially humans. Vaccines of the present invention can be prophylactic(e.g. to prevent or ameliorate the effects of a future infection by anynatural or “wild” pathogen), or therapeutic (e.g. vaccines againstcancer).

Vaccination is the most important route of dealing with viralinfections. Although a number of antiviral agents are available,typically these agents have limited efficacy. Administering antibodiesagainst a virus may be a good way of dealing with viral infections oncean individual is infected (passive immunization) and typically human orhumanized antibodies do seem promising for dealing with a number ofviral infections. But the most efficacious and safe way of dealing withvirus infection is, and probably will be, prophylaxis through activeimmunizations. Active immunization is generally referred to asvaccination and vaccines comprising at least one antigenic determinantof a virus, preferably a number of different antigenic determinants ofat least one virus, e.g., by incorporating in the vaccine at least oneviral polypeptide or protein derived from a virus (subunit vaccines).Typically, the formats mentioned so far include adjuvants in order toenhance an immune response. This also is possible for vaccines based onwhole virus, e.g., in an inactivated format. A further possibility isthe use of live, but attenuated forms of the pathogenic virus. A furtherpossibility is the use of wild-type virus, e.g., in cases where adultindividuals are not in danger from infection, but infants are and may beprotected through maternal antibodies and the like.

Production of vaccines is not always an easy procedure. In some casesthe production of viral material is on eggs, which leads to difficultyin purifying material and extensive safety measures againstcontamination, etc. Also production on bacteria and or yeasts, whichsometimes, but not always, is an alternative for eggs, requires manypurification and safety steps. Production on mammalian cells would be analternative, but mammalian cells used so far all require, for instance,the presence of serum and/or adherence to a solid support for growth. Inthe first case, again, purification and safety and e.g., the requirementof protease to support the replication of some viruses become an issue.In the second case, high yields and ease of production become a furtherissue.

Vaccines are still lacking for many viral diseases of great publichealth importance. Killed viral vaccines can be dangerous and expensiveto produce, and are frequently not immunogenic. The inclusion of viralprotein encoding sequences in an Adenoviral vector may circumvent theseproblems, however, there are challenges to creating such an Adenovirusvector. For example, there is little space in Adenovirus vectors, andthe immune response to the Adenovirus vector interferes with the immuneresponse to the vaccine protein.

An object of the present invention is therefore to provide GDVs, whichare capable of a long-term maintenance in a large and increasing numberof different cells of the host's body and thereby capable of providing astable expression of the desired antigen(s). Another object of theinvention is to provide GDVs, which are maintained for a long period oftime in the cells that originally received the vector and transferred itto the daughter cells after mitotic cell division. Yet another object ofthe invention is to provide GDVs, which express in addition to the geneor genes of interest preferably only a gene necessary for a long-termmaintenance in the recipient cells and thus are devoid of componentsthat are toxic or cause symptoms of the disease to the recipient. Afurther object of the invention is to provide GDVs, which mimicattenuated live viral vaccines, especially in their function ofmultiplying in the body, without inducing any considerable signs ofdisease and without expressing undesired proteins, which may induceadverse reactions in a host injected with the DNA vaccine. The vaccinesof the present invention comprise a GDV of the present invention or amixture of said vectors in a suitable pharmaceutical carrier. Thevaccines of the invention are formulated using standard methods ofvaccine formulation to produce vaccines to be administered by anyconventional route of administration, i.e. intramuscularily,intradermally and like. In specific embodiments, a GDV of the inventionis used to treat and/or prevent an infectious disease and/or a conditioncaused by an infectious agent. Such diseases and conditions include, butare not limited to, infectious diseases caused by bacteria, viruses,fungi, protozoa, helminths, and the like.

A GDV of the present disclosure can be used for vaccine development toprotect an individual against a disease by inducing immunity. Oneadvantage of using a GDV of the present disclosure for vaccinedevelopment is that the recipient's immune response is not deflected byAd genes. In an embodiment, a GDV of the present disclosure encodes oneor more proteins and/or RNAs (angitens) from viruses of importance forhuman health or agriculture. In an embodiment, the vaccine is used toprotect an individual against a disease by inducing immunity. In anembodiment, multiple genes of interest my be included for mulitivalentvaccines, from the same or different pathogens. In certain embodiments,the GDV further comprises one or more expression cassettes of a DNAsequence of interest. In certain embodiments, the DNA sequence ofinterest is that of an infectious pathogen. In certain embodiments, theinfectious pathogen is a virus. In certain specific embodiments, thevirus is selected from the group consisting of Human ImmunodeficiencyVirus (HIV), Herpex Simplex Virus (HSV), Hepatitis C Virus, InfluenzaeVirus, Rotavirus, Papilloma Virus, Lentivirus, Enterovirus orcombinations thereof. In certain embodiments, the DNA sequence ofinterest is that of a bacterium. In certain embodiments, the bacteriumis selected from the group consisting of Chlamydia trachomatis,Mycobacterium tuberculosis, and Mycoplasma pneumonia. In certainembodiments, the DNA sequence of interest is that of a fungal pathogen.In certain embodiments, the DNA sequence of interest is of HIV origin.In an embodiment, the vaccine is used to protect an individual againstinfluenza virus. In an embodiment, the influenza virus is swine flu. Inan embodiment, the influenza virus is avian flu. In an embodiment, theone or more proteins and/or RNAs of a GDV of the present disclosure areselected from one of hemagglutinin (HA), neuraminidase (NA),nucleocapsid (NP), M₁ (matrix protein), M₂ (ion channel), NS₁, NS₂(NEP), lipid bilayer, PB1, PB2 or PA.

Representative viral and bacterial candidates for vaccines of thepresent disclosure are listed below. Genes for these vaccines areillustrated in italic.

Genetic Adjuvants

-   interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4    and IL-10)-   GM-CSF

Viruses—Orthomyxoviruses

-   Influenza A: hemagglutinin (HA) and neuraminidase (NA),    nucleoprotein (NP), M₁, M₂, NS1, NS2 (NEP), PA, PB1, PB1-F2 and PB2)-   Influenza B-   Influenza C

Viruses—Herpes Virus

-   Herpes simplex 1 (oral herpes)-   Herpes simplex 2 (genital herpes)—84 polypeptides; 11 viral    glycoproteins (designated gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL,    and gM) are known, and another is (gN) predicted; glycoproteins B    and D.-   Epstein Barr (mononucleosis, Burkitt's lymphoma, nasopharyngeal    carcinoma)—Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP,    and LMP; gp350/220 aka gp340-   Cytomegalovirus—Glycoprotein B, IE1, pp89, gB and pp65 are the    minimum requirements in a vaccine to induce neutralising antibodies    and cytotoxic T-lymphocyte (CTL) responses. Immunisation with    additional proteins, e.g., gH, gN for neutralising antibodies and    IE1exon 4 and pp150 for CTL responses, would strengthen protective    immune responses.-   Varicella zoster virus (chicken pox and shingles)—recombinant    proteins from gE, gI and gB genes-   Kaposi's sarcoma-associated herpesvirus 8 (Kaposi's sarcoma)-   Herpes 6 (A and B)-   Herpes 7-   Herpes B: glycoprotein B (gB);-   Viruses—Papilloma virus: For all HPV L1 capsid protein, E1, E2, E6,    and E7 genes HPV (Cervical carcinoma High-risk: 16, 18, 31, 33, 35,    39, 45, 51, 52, 56, 58, 59;-   Probably high-risk: 26, 53, 66, 68, 73, 82)-   HPV (common warts: 2, 7)-   HPV (Plantar warts: 1, 2, 4, 63)-   HPV (Flat warts: 3, 10)-   HPV (Anogenital warts: 6, 11, 42, 43, 44, 55)

Viruses—Reoviridae

-   Rotavirus A (gastroenteritis)—VP2 and VP6 proteins

Viruses—Coronaviruses

-   Severe acute respiratory syndrome coronavirus (Severe Acute    Respiratory Syndrome)—SARS-CoV is an enveloped plus-stranded RNA    virus with a ˜30 kb genome encoding replicase (Rep) and the    structural proteins spike (S), envelope (E), membrane (M), and    nucleocapsid (N) spike or nucleocapsid proteins, S and N genes    respectively-   Human coronavirus 229E—spike and envelope genes-   Human Coronavirus NL63-   Viruses—Astrovirus (gastroenteritis)—the astrovirus 87-kDa    structural polyprotein-   Viruses—Norovirus (gastroenteritis)—Viral capsid genes, VP1 and VP2

Viruses—Flaviviridae

-   Dengue fever—premembrane (prM) and envelope (E) genes-   Japanese encephalitis—prM, E and NS1 genes; prM, and envelope (E)    coding regions of JE virus.-   Kyasanur Forest disease-   Murray Valley encephalitis-   St. Louis encephalitis-   Tick-borne encephalitis-   West Nile encephalitis-   Yellow fever-   Hepatitis C—Hepatitis C Virus Glycoprotein E2; glycoproteins E1 and    E2 of hepatitis C; The core gene of HCV

Viruses—Picornaviridae—Enterovirus

-   Human enterovirus A (21 types including some coxsackie A viruses)-   Human enterovirus B (57 types including enteroviruses, coxsackie B    viruses)-   Human enterovirus C (14 types including some coxsackie A viruses)-   Human enterovirus D (three types: EV-68, EV-70, EV-94)—VPI gene;

Viruses—Picornaviridae—Rhinovirus

-   Human rhinovirus A (74 serotypes)-   Human rhinovirus B (25 serotypes)-   Human rhinovirus C (7 serotypes)—rhinovirus-derived VP1; the surface    protein which is critically involved in infection of respiratory    cells

Viruses—Picornaviridae—Hepatovirus

-   Hepatitis A

Viruses—Togaviridae—Alphavirus

-   Sindbis virus-   Eastern equine encephalitis virus-   Western equine encephalitis virus-   Venezuelan equine encephalitis virus-   Ross River virus-   O'nyong'nyong virus

Viruses—Togaviridae—Rubivirus

-   Rubella virus

Viruses—Togaviridae—Hepevirus

-   Hepatitis E virus—the ORF2 protein; recombinant HEV capsid protein;    the vaccine peptide has a 26 amino acids extension from the N    terminal of another peptide, E2, of the HEV capsid protein

Viruses—Togaviridae—Bornaviridae

-   Borna disease virus—BDV nucleoprotein (BDV-N)

Viruses—Togaviridae—Filoviridae

-   Ebolavirus-   Marburgvirus

Viruses—Togaviridae—Paramyxoviruses

-   Measles-   Sendai virus-   Human parainfluenza viruses 1 and 3,-   Mumps virus-   Human parainfluenza viruses 2 and 4-   Human respiratory syncytial virus-   Newcastle disease virus

Viruses—Togaviridae—Retrovirus

-   HIV—gag: p18, p24, p55; pol: p31,p51,p66; env: p41, p120, p160-   hepatitis B virus

HTLV I, II

Viruses—Togaviridae—Rhabdoviruses

-   Rabies

Viruses—Togaviridae—Arenaviruses

-   Hanta virus-   Korean hemorrhagic fever-   Lymphocytic choriomeningitis virus-   Junin-   Machupo-   Lassa-   Sabia-   Guanarito-   California encephalitis-   Congo-Crimean hemorrhagic fever-   Rift valley fever

Viruses—Parvoviruses

-   Human parvovirus (B19)

Bacteria

-   Bartonella-   Brucella including B. abortus, B. canis, B. suis-   Burkholderia (Pseudomonas) mallei, B. pseudomallei-   Coxiella burnetii-   Francisella tularensis-   Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk    Group 2 (RG2)—Bacterial Agents Including Chlamydia), M. tuberculosis-   Pasteurella multocida type B -“buffalo” and other virulent strains-   Rickettsia akari, R. australis, R. canada, R. conorii, R.    prowazekii, R. rickettsii, R. siberica, R. tsutsugamushi, R. typhi    (R. mooseri)-   Yersinia pestis

A method of regulating an immune response in an individual includesadministering a GDV of the present disclosure to the individual, whereinthe gene transfer vector encodes at least one therapeutic gene (gene ofinterest, “GOI”).

The present invention is described in the following Examples, which areset forth to aid in the understanding of the invention, and should notbe construed to limit in any way the scope of the invention as definedin the claims which follow thereafter. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the describedinvention, and are not intended to limit the scope of what the inventorsregard as their invention nor are they intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (e.g.amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, temperatureis in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLES Example 1 Construction of a Packaging Nucleic Acid Constuct(pPack) (FIG. 7)

As illustrated in FIG. 7, Adenoviral late genes were introduced into apBR322 vector by the sequential cloning of specific fragments of Ad5DNA. A polylinker was introduced into pBR322 by ligating pBR322 cut withthe restriction enzymes Hind III and Sal 1 and a polylinker composed ofthe complementary nucleic acids:

(SEQ ID NO: 1) AGCTAACTATCCCATTAATTAACCGTCCATTTTCGAAAATGCTACCCGGGAATACGTTACGTATGAATCTGGCGCGCCTAACGTAGGATCCAATGCTAACTAGTATAAGATATTTAAATAAGCCCA and (SEQ ID NO: 2)TCGATGGGCTTATTTAAATATCTTATACTAGTTAGCATTGGATCCTACGTTAGGCGCGCCAGATTCATACGTAACGTATTCCCGGGTAGCATTTTCGAAAATGGACGGTTAATTAATGGGATAGTT.

The ligation was then transformed into chemically competent E. coli DH5alpha (InVitrogen) and ampicillin resistant colonies were selected. Thepolylinker contained sites for the following restriction enzyme cleavagesites (in order): Bst B1, Xma, Sna B1, Asc 1, Bam H1, Spe 1, and Swa 1.The following Ad5 DNA fragments were cloned into this vector in order:Bst B1 (4071)—Xma (6737); Xma (6737)—Sna B1 (10,461); Sna B1(10,461)—Asc I (15,823); Asc I (15,823)—Bam H1 (21,714); Bam H1(21,714)—Spe1 (27,234); Spe1 (27,234)—Sna B1. The last fragment wascloned into the polylinker between the Spe 1 and Swa 1 sites.

Example 2 Construction of a Packaging Nucleic Acid Construct (pPack)(FIG. 3)

As illustrated in FIG. 3, a recombination plasmid carrying the SV40origin of replication and early region as well as left and right arms ofthe Ad genome was constructed. pBR322 was cleaved with restrictionenzymes Eco R1 and Bam H1 and ligated with a polylinker composed of thecomplementary oligonucleotides: 5′ G ATC CCT ACG GTA CCT ACG TCT AGA CAGTG 3′ (SEQ ID NO: 3) and 5′ AAT TCA CTG TCT AGA CGT AGG TAC CGT AGG 3′(SEQ ID NO: 4). The ligation was transformed into chemically competentDH5alpha E. coli (InVitrogen) and colonies resistant to ampicillin wereselected. In addition to restoring the Eco R1 and Bam H1 sites, thispolylinker introduced unique sites for the restriction enzymes Kpn I andXba I, such that the order of sites was: Bam H1, Kpn 1, Xba 1, and EcoR1. Three fragments were then cloned into this vector at sites in thepolylinker.

The SV40 origin of replication and Early region was cloned as a BamH1/Kpn fragment (nt 2533 to nt 5243/0, continuing to nt 298) at the Kpnand Bam H1 sites in the polylinker. A fragment of the left arm of Ad5genome from nt 4501 to nt 5466, was amplified by PCR from Ad5 DNA usingthe primers: 5′ GGT ACC TGT ATC CGG TGC ACT TGG GAA ATT TG 3′ (SEQ IDNO: 5) and 5′ TCT AGA ACA CCA TGG TCA AAT GCT ACC TGG G 3′ (SEQ ID NO:6). The resulting DNA fragment was cleaved with restriction enzymes Kpn1 and Xba 1 and ligated between the Kpn and Xba 1 sites in thepolylinker. A fragment of the right arm, from nt 34,677 to nt 35836,just before the right ITR sequence was amplified by PCR, cleaved withthe restriction enzymes Eco R1 and Xba 1 and ligated into the polylinkerat the Xba 1 and Eco R1 sites.

This plasmid was recombined with the Ad 5 genomic DNA in E. coli. Theplasmid was digested with the restriction enzyme Xba to produce a linearmolecule with the regions of Ad5 left and right arms at the two ends ofthe linearized plasmid. The linear plasmid and wild-type Ad5 genomic DNAwere co-electroprated into rec A⁺ E. coli and ampicillin resistantcolonies selected. Recombination between the sequences at the ends ofthe linearized plasmids and the Ad5 genomic DNA resulted in a circularplasmid containing an ampicillin resistance gene, a bacterial origin ofreplication, the SV40 origin of replication and Early region, and the Adlate genes but without the IX and E1 genes, the Ad packaging site, oreither of the Ad ITRs.

Example 3 Construction of a Fully-Deleted Adenoviral Vector (FDV)Carrying the H1N1 Influenza Hemagglutinin Gene (FIG. 1)

The “stiffer” basis for this FDV was a bacterial artificial chromosome(BAC) vector carrying the human ATIC gene encoding5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMPcyclohydrolase. A targeting plasmid construct for transforming thishuman BAC ATIC clone into a FDV was composed of: the left Ad ITR and Ψ;the right Ad ITR; the gene of interest, in this case the H1N1 influenzaneuraminidase in an expression cassette with a cytomegalovirus promoterat the five prime end and a polyadenylation sequence at the three primeend; two regions from the human ATIC gene to mediate homologousrecombination with the human ATIC BAC; and a plasmid vector with abacterial origin of replication and an antibiotic resistance gene.

The arrangement of these elements is schematically shown in FIG. 1. TheAd left ITR and Ψ sequence are amplified by PCR from Ad 5 genomic DNAwith two primers:

(SEQ ID NO: 7) AATACCCGGGAATATGAGCTCATCATGTTTAAACAATCATCATCAATAATATACCTATTTTG  and (SEQ ID NO: 8)ACATATCTAGAAACAGTCTCCACGTAAACGGTCAAAG.

The right ITR was amplified by PCR from Ad 5 genomic DNA using twoprimers:

(SEQ ID NO: 9) TAACATGCATAATATGCCCGGGCATAGCGGCAGCCTAACAGTCAGCC TTACC and(SEQ ID NO: 10) AATGGGCCCATATAGTTTAAACATACATCATCAATAATATACCTATT TTG.The vector was pBR322.

The two regions of human ATIC DNA, from exons 3 and 16, were amplifiedby PCR using two pairs of primers:

exon 3  (ATAAGCTTGAACATACACAACAGTTAGG (SEQ ID NO: 11) andATAAGCTTTCTGGCAGCAACTTCATAAC) (SEQ ID NO: 12) and exon 16(TTATCGATGATGAAGATTTGATAAAGTGG (SEQ ID NO: 13) andTTGGCGCGCCGTTATCCTCAAAGTTTCAGGC). (SEQ ID NO: 14)

The neuraminidase gene was amplified by PCR from cDNA isolated fromcells infected by the influenza (A/California/07/2009(H1N1)). The PCRprimers were:

(SEQ ID NO: 15) CTGTCGACAAATGAATCCAAACCAAAAGAT  and (SEQ ID NO: 16)TCGAAGCTTAGTTACTTGTCAATGGTAAATG.

The targeting vector composed of these sequences was linearized bycleavage with a restriction endonuclease that cuts the plasmid uniquelybetween the ATIC regions. This linearized plasmid and the human ATIC BACwere co-electroporated into recombination proficient E. coli, andampicillin resistant colonies were selected. The plasmid resulting fromhomologous recombination between these two molecules was the FDV. Forsubsequent use, this FDV carrying the H1N1 neuraminidase gene wasliberated by cleaving with a restriction endonuclease that cleaves atthe outer edges of the left and right ITRs.

Example 4 Construction of An Adenoviral Packaging Cell (PC) LineCarrying the Ad5 E1 and IX Genes by Cell Transformation (FIG. 8)

As illustrated in FIG. 8, a DNA fragment containing the Ad5 E1 and IXgenes was produced by polymerase chain reaction (PCR) using Ad5 genomicDNA as a template and primers: AAT ACT CGA GAT AAT GAA TTC ATA TCG CCCAGG TGT TTT TCT CAG G (SEQ ID NO: 17) and AAT AGG ATC CAT AAT GAA TTCATA GAT CCA AAT CCA AAC AGA GTC (SEQ ID NO: 18). This amplified the Ad5region from nt 385 to nt 4070. PCR cycling conditions were thirtycycles: 95° C., 1 min; 60° C., 2 min; and 68° C., 4 min.

A549 cells were grown to 90% confluency in a T-75 tissue culture flaskwith serum free RPMI (RPMI SF), Sigma R6504. On the day beforetransfection, the media was removed from the flask via aspiration, andthe cells were detached by adding 5 ml 0.25% trypsin/EDTA (Sigma T3449)to the flask. After 5 minutes of incubation at 37° C., the solution withthe cells was centrifuged for 5 min at 200×g with 10 ml RPMI in a 50 mlconical tube in a swinging rotor centrifuge (Centra CLR3). Afterdecanting the supernatant, the cells were resuspended in 5 ml RPMI SFand pipetted up and down seven times gently to attain a single cellsuspension. After quantitating the viable cells by trypan blue stainingand counting in a hemacytometer, 1×106 cells were plated in each well ofa 6 well tissue culture plate with 5 ml RPMI SF and swirled gently toevenly distribute the cells. The cells were incubated overnight intissue culture incubator.

On the day of transfection, the media was removed by aspiration andimmediately replaced with fresh RPMI SF (2 ml). To prepare the DNA fortransfection, 12 μg of the E1/DC DNA was added to 400 μl 150 mM sterileNaCl, and then 24 μl jetPEI™ (PolyPlus 101-10) was added to 400 μl 150mM sterile NaCl. DNA/NaCl mixture was slowly added to the jetPEI™/NaClmixture. The solution was incubated for 20 minutes at room temperature.200 μl of the DNA/jetPEI™ solution was added to each well. The cellswere then incubated overnight in a tissue culture incubator.

The day after transfection, the medium was removed from each well viaaspiration and 5 ml RPMI SF with 1.5% carboxymethyl-celluose (SigmaC5013) was added. The cultures were maintained in the tissue cultureincubator and the medium was changed twice per week. The well wasexamined daily for proliferation beyond a monolayer of cells, that is,for cell colonies that had lost contact inhibition and were formingdiscernable masses above the monolayer. These colonies appear from 10 to90 days after transfection.

When colonies are of sufficient size (>100 cells), they were removedwith a sterile pipet tip and seeded on a single well in a 6 well plate(5 ml RPMI SF, without methyl-cellulose). The single colony cultureswere grown until 90% confluence. They were then passaged by standardtechniques using the same medium.

DNA was isolated from these single colony cultures using standard DNAisolation kits such as DNeasy (Qiagen 69504), and was then tested forthe presence of the E1/IX DNA fragment by PCR using the conditionsdescribed above. The expression of the E1 and IX genes was then testedby RT-PCR using primer pairs for E1 (AT GAG ACA TAT TAT CTG CCA CGG (SEQID NO: 19) (nt 560-nt 582) and TTA TGG CCT GGG GCG TTT ACA G (SEQ ID NO:20) (nt 1524-nt 1545) and for the IX gene (AT GAG CAC CAA CTC GTT TGA TG(SEQ ID NO: 21) (nt 3609-nt 3630) and TT AAA CCG CAT TGG GAG GGG (SEQ IDNO: 22) (nt 4012-nt 4030).

Example 5 Construction of An Adenoviral Packaging Cell (PC) LineCarrying the Ad5 E1 and IX Genes by Co Transfection with pSV2-Neo (FIG.9)

As schematically illustrated in FIG. 9, A549 cells were grown to 90%confluence in a T-75 tissue culture flask with serum free RPMI (RPMISF), Sigma R6504. On the day before transfection, the media was removedfrom the flask via aspiration, and the cells were detached by adding 5ml 0.25% trypsin/EDTA (Sigma T3449) to the flask. After 5 minutes ofincubation at 37° C., the solution with the cells, was centrifuged for 5min at 200×g with 10 ml RPMI in a 50 ml conical tube in a swinging rotorcentrifuge (Centra CLR3). After decanting the supernatant, the cells were resuspended in 5 ml RPMI SF and pipetted up and down seven timesgently to attain a single cell suspension. After quantitating the viablecells by trypan blue staining and counting in a hemacytometer, 1×106cells were plated in each well of a 6 well tissue culture plate with 5ml RPMI SF and swirled gently to evenly distribute the cells. The cellswere incubated overnight in tissue culture incubator.

On the day of transfection, the media was removed by aspiration andimmediately replaced with fresh RPMI SF (2 ml). To prepare the DNA fortransfection, 6 μg of the E1/IX DNA was added and 6 μg of PSV2Neo DNA to400 μl 150 mM sterile NaCl, and then 24 μl jetPEI™ (PolyPlus 101-10) wasadded to 400 μl 150 mM sterile NaCl. The DNA/NaCl mixture was slowlyadded to the jetPEI™/NaCl mixture. The solution was incubated for 20minutes at room temperature. 200 μl of the DNA/jetPEI™ solution wasadded to each well. The cells were then incubated overnight in a tissueculture incubator.

Forty-eight hours after transfection, the medium was removed from thewell via aspiration and 5 ml RPMI with 1.0 mg per ml G418 (geneticin)was added. The cultures were maintained in the tissue culture incubatorand medium with G418 was changed daily. The well was examined daily fordeath of non-transfected cells and proliferation of transfected cells.Non-adherent dead cells were removed via medium changes.

When sufficient numbers of cells were present in a geneticin resistantcolony (>1000 cells), they were subcloned as single colonies. The singlecolony cultures were grown until 90% confluence. They were then passagedby standard techniques using the same medium.

DNA was isolated from these single colony cultures using standard DNAisolation kits such as DNeasy (Qiagen 69504), and was then tested forthe presence of the E1/IX DNA fragment by PCR using the conditionsdescribed above. The expression of the E1 and IX genes was then testedby RT-PCR using primer pairs for E1 (AT GAG ACA TAT TAT CTG CCA CGG (SEQID NO: 19) (nt 560-nt 582) and TTA TGG CCT GGG GCG TTT ACA G (SEQ ID NO:20) (nt 1524-nt 1545) and for the IX gene (AT GAG CAC CAA CTC GTT TGA TG(SEQ ID NO: 21) (nt 3609-nt 3630) and TT AAA CCG CAT TGG GAG GGG (SEQ IDNO: 22) (nt 4012-nt 4030).

Example 6 Packaging of a FDV Carrying the Human Factor VIII CodingSequence by Co-Transfection with a Packaging Construct (pPack) toPropagate GDVs (FIG. 10)

QBI293A cells were grown to 90% confluence in a T-75 tissue cultureflask with serum free RPMI 10% FCS (RPMI), Sigma R6504. On the daybefore transfection, media was removed from the flask via aspiration,and cells were detached by adding 5 ml 0.25% trypsin/EDTA (Sigma T3449)to the flask. After 5 minutes of incubation at 37° C., the solution withthe cells was centrifuged for 5 min at 200×g with 10 ml RPMI in a 50 mlconical tube using a swinging rotor centrifuge (Centra CLR3). Afterdecanting the supernatant, cells were resuspended in 5 ml RPMI SF andpipetted up and down seven times gently to attain a single cellsuspension. After quantitating the viable cells by trypan blue stainingand counting in a hemacytometer, 1×10⁶ cells were plated in each well ofa 6 well tissue culture plate with 5 ml RPMI and swirled gently toevenly distribute the cells. The cells were incubated overnight in atissue culture incubator.

On the day of transfection, the media was removed by aspiration andimmediately replaced with fresh RPMI (2 ml). To prepare the DNA forco-transfection, 4 μg of DNA of a fully-deleted Ad vector carryingFactor VIII and 4 μg of DNA from a plasmid carrying the Ad 5 sequences(nt 3534-28,129, and nt 30,821-35931) were added to 400 μl 150 mMsterile NaCl, and then 24 μl jetPEI™ was added (PolyPlus 101-10) to 400μl 150 mM sterile NaCl. The DNA/NaCl mixture was added slowly to thejetPEI™/NaCl mixture. The solution was incubated for 20 minutes at roomtemperature. Next, 200 μl of the DNA/jetPEI™ solution was added to eachwell. The cells were incubated for four hours in a tissue cultureincubator. After incubation, the medium was replaced with 5 ml RPMI andthe cells were returned to a tissue culture incubator.

Forty-eight hours after transfection the cells were scraped from thesurface of each well using a cell scraper (Falcon 353086). The solutionwas transferred to a 50 ml conical tube, combining like wells. The cellsuspension was centrifuged for five minutes at 200×g and supernatant wasdecanted without disturbing the cell pellet. The pellet was resuspendedin 100 μl RPMI. The pellet was frozen in a dry ice/isopropanol bath forthree minutes and was then thawed in a 37° C. water bath for threeminutes. The freeze thaw cycle was repeated a total of three times. Theresultant cell lysate was centrifuged for eight minutes at 1200×g. Thesupernatant was removed with a micropipet and diluted 10 times withRPMI. The supernatant, GDV Factor VIII preparation, was stored at −80°until infection.

The GDV Factor VIII preparation was analyzed by quantitating virions bya sandwich ELISA using a monoclonal anti-body against Ad5 virions (FIG.10A). This clearly showed the presence of Ad virions at only slightlylower concentration than the positive control, a first generation Advector carrying the mouse CD8. To demonstrate the infectivity of the GDVFactor VIII preparation, A549 human fibroblasts were infected with theGDV Factor VIII preparation. 48 hours after infection, the cells wereharvested for DNA and the isolated DNA was analyzed by PCR for F8 usingprimers specific for the F8 transgene (Forward primer from the CMVpromoter: Cgcgttacataacttacggta (SEQ ID NO: 23) ; Reverse primer fromthe Factor VIII coding sequence: ccagggaagactttatcatc (SEQ ID NO: 23))(FIG. 10B). DNA from cells infected with GDV Factor VIII gave a positivePCR signal, while uninfected cells did not. This showed the transductionof the GDV Factor VIII DNA into cells infected with the GDV Factor VIII.

To demonstrate the secretion of F8 from cells infected with GDV FactorVIII, an EliSpot assay for F8 secreting cells was used. An Elispot plate(Millipore MSIPS4W10) was wetted with 15 μl/well 35% sterile ethanol.This was followed with three 150 μl sterile PBS washes. F8 captureantibody (Abcam 53203) was added at a 1 to 5000 dilution, 100 μl/well inPBS. The plate was incubated overnight at 4° C. The plate was washed twotimes with 150 μl PBS/well, and was then blocked overnight with 3% BSAin PBS 150 μl/well.

A549 cells grown to 90% confluence in a T-75 flask were trypsinized andresuspended, and the cells were counted using a hemacytometer. The cellswere seeded on an Elispot plate at 50 thousand cells/well in a volume of50 μl RPMI/well. The cells were incubated overnight in a tissue cultureincubator. On the following day, 50 μl of GDV Factor VIII supernatant(above) was added to each well of cells. The cells were held overnightin tissue culture incubator.

Plate development began with five washes of 150 μl/well of PBS /0.01%Tween-20 and two washes 150 μl PBS to remove cells. F8-HRP conjugatedantibody (American Diagnostica ESH-8R) at 1 to 4000 dilution in PBS 0.5%BSA was added to each well, and the plate was incubated for 1.5 hours at37° C. The plate was then washed seven times with 200 μl PBS/well. Thesubstrate was prepared by dissolving 4.0 mg 3-Amino 9ethyl carbazole(Sigma 038K1032) in 1 ml dimethyl formamide (Sigma D-4451), which wasthen added to 14 ml citrate-phosphate buffer, 0.1M pH 6.0, and 15 μl 30%H₂O₂. 100 μl of the substrate solution was added to each well, incubatedfor ten minutes at room temperature and then washed liberally with tapwater. Cells infected with GDV Factor VIII gave positive signal in theEliSpot assay indicating their secretion of F8 (FIG. 10C).

Example 7 Keratinocyte and Fibroblast Isolation, Characterization,Engineering and Transplantation

As a prelude to establishing a skin transplantation model, keratinocytesand fibroblasts were isolated from neonatal and adult mice using thefollowing protocol:

Day old neonatal mouse pups were sacrificed by decapitation then alllimbs were removed. The body was immersed in providone/iodine for 2minutes then rinsed in 70% ethanol for 2 more minutes, followed by 2more minutes in PBS/PSN. The skin was surgically removed by first makinga ventral incision anterior to posterior then, the skin was gentlypeeled off in one piece. Skins were rinsed briefly in PBS/PSN thenplaced in 5 mls Dispase, (BD) at 4° C. for 16 hours. After Dispasetreatment the skins were rinsed in PBS/PSN and transferred to a 10 cmPetri dish with 10 mls PBS/PSN. The epidermis was gently peeled off thedermis in one continuous sheet. The epidermal sheets were floated on 1ml drops of trypsin at room temp for 15 to 20 minutes. The epidermalsheets were then gently agitated with forceps to release keratinocyteclumps. 10 mls of PBS/PSN 10% FCS was added and the skins were pipettedup and down with a 25 ml pipette to further release keratinocytes. Thevolume was brought to 50 mls with PBS/PSN 10% FCS and filtered through a100 μm cell strainer. The cells were pelleted at 300×g for 4 minutes,washed with PBS/PSN 10% FCS, resuspended in growth medium, and counted.

After step six above, the dermis was collected and briefly rinsed inPBS/PSN. The dermis was incubated for 15 to 20 minutes in 10 mls of 1mg/ml collagenase type V, (Sigma) in PBS, with gentle inversion at 37°C. The collagenase was neutralized with the addition of 20 mls PBS/PSN10% FCS. The cell clumps were dissociated with gentle pipetting using a25 ml pipette. The volume was brought to 50 mls with PBS/PSN 10% FCS andthe cells were filtered through a 100 μm cell strainer. The cells werepelleted at 300×g for 5 minutes, washed with PBS/PSN 10% FCS,resuspended in growth media and counted.

Keratinocytes and fibroblasts harvested from both neonates and adultswere then analyzed by FACS analysis both for the expression of MHC classI and class II expression, and for tissue specific markers.Representative data from adult keratinocytes are presented in Table 5.

TABLE 5 FACS analysis of keratinocytes harvested from adult mice.Numbers are % positive cells. Surface Antigen MHC I MHC II CD98 Integrin6 epCAM Positive [%] 84.5 5.6 95.4 88.4 0.7

In contrast, neither neonatal fibroblasts nor keratinocytes expressedhigh levels of MHC or differentiation markers (Table 6). Prolongedincubation of neonatal keratinocytes and fibroblasts lead to increasedexpression of both MHC and tissue specific antigens.

TABLE 6 FACS analysis of keratinocytes and fibroblasts from neonatalmice. Surface Antigen - [%] Positive MHC I MHC II CD98 Integrin 6 epCAMKeratinocytes 0.6 0.5 67 31.7 0.1 Fibroblastsz 0.7 1.7 91.1 2.3 0

As reported in the literature, keratinocytes showed different growthrequirements. While keratinocytes required specialty media (e.g. Cnt o2from Millipore), fibroblasts grew well on standard Eagle's medium. Thisis an important consideration when producing engineered skin containingboth keratinocytes and fibroblasts.

We used three different matrices to produce engineered skin:decellularized mouse dermis, Neomem membrane (Citagenix), andTranswell-col (Corning International Life Sciences). In all cases,fibroblasts were “pre-seeded” on the membrane, before the keratinocyteswere, and the membranes were incubated in medium especially designed tofoster growth of keratinocytes on matrices (Con02-3D, Millipore). Bothkeratinocytes and fibroblasts will be infected with a GDV of the presentdisclosure that is based on Human Serotype 5, carrying CD8, andpreferably the human CD8α-chain. In an embodiment, the viral capsid canbe altered (pseudotyped) in the hypervariable region of the viral hexon.Infection efficiency will be determined by FACS analysis for cellsurface expression of CD8.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for propagating a fully-deletedadenovirus-based gene transfer vector comprising: (a) providing anAdenovirus packaging cell line; (b) transfecting, into the cell line, afully-deleted Adenoviral vector construct; and (c) transfecting, intothe cell line, a replication defective packaging construct having asubset of Adenoviral late genes while being absent of at least oneinverted terminal repeat and a packaging signal, wherein thefully-deleted Adenoviral vector construct and the packaging constructcan transfect the Adenovirus packaging cell line resulting in theencapsidation of a fully-deleted adenoviral-based gene transfer vectorindependent of helper Adenovirus, wherein the encapsidated fully-deletedadenovirus-based gene transfer vector includes both adenoviral invertedterminal repeats, the packaging signal, and at least one DNA insertwhich comprises a gene sequence encoding a protein of interest, andwherein the encapsidated fully-deleted adenovirus-based gene transfervector is absent of adenoviral structural genes.
 2. The method of claim1 wherein the Adenovirus packaging cell line is a human cell line. 3.The method of claim 2 wherein the human cell line is an A549 cell line.4. The method of claim 2 wherein the human cell line is derived from aprimary culture.
 5. The method of claim 2 wherein the human cell line isa HeLa cell line.
 6. The method of claim 1 wherein the Adenoviruspackaging cell line comprises an Adenovirus early region 1 (E1) codingsequence.
 7. The method of claim 1 wherein the fully-deleted Adenoviralvector construct comprises at least one inverted terminal repeat, apackaging signal, at least one gene expression construct, and humangenomic stuffer DNA.
 8. The method of claim 1 wherein the fully-deletedAdenoviral vector construct comprises a subset of Adenoviral late genes.9. The method of claim 1 wherein the replication defective packagingconstruct itself is incapable of being packaged.
 10. The method of claim1 wherein the replication defective packaging construct includes asubset of Adenoviral late genes selected from the group consisting ofL1, L2, L3, L4, L5, E2A and E4.
 11. The method of claim 1 wherein theencapsidated fully-deleted adenovirus-based gene transfer vector isreplication deficient.
 12. A method for propagating a fully-deletedadenovirus-based gene transfer vector comprising: (a) providing anAdenovirus packaging cell line, wherein the Adenovirus packaging cellline is a human cell line; (b) transfecting, into the cell line, afully-deleted Adenoviral vector construct; and (c) transfecting, intothe cell line, a replication defective packaging construct that isincapable of being packaged, the replication defective packagingconstruct having a subset of Adenoviral late genes while being absent ofat least one inverted terminal repeat and a packaging signal, whereinthe fully-deleted Adenoviral vector construct and the packagingconstruct can transfect the Adenovirus packaging cell line resulting inthe encapsidation of a fully-deleted adenoviral-based gene transfervector independent of helper Adenovirus, wherein the encapsidatedfully-deleted adenovirus-based gene transfer vector includes bothadenoviral inverted terminal repeats, the packaging signal, and at leastone DNA insert which comprises a gene sequence encoding a protein ofinterest, and wherein the encapsidated fully-deleted adenovirus-basedgene transfer vector is absent of adenoviral structural genes.
 13. Themethod of claim 12 wherein the human cell line is an A549 cell line. 14.The method of claim 12 wherein the human cell line is derived from aprimary culture.
 15. The method of claim 12 wherein the human cell lineis a HeLa cell line.
 16. The method of claim 12 wherein the Adenoviruspackaging cell line comprises an Adenovirus early region 1 (E1) codingsequence.
 17. The method of claim 12 wherein the fully-deletedAdenoviral vector construct comprises at least one inverted terminalrepeat, a packaging signal, at least one gene expression construct, andhuman genomic stuffer DNA.
 18. The method of claim 12 wherein thefully-deleted Adenoviral vector construct comprises a subset ofAdenoviral late genes.
 19. The method of claim 12 wherein thereplication defective packaging construct includes a subset ofAdenoviral late genes selected from the group consisting of L1, L2, L3,L4, L5, E2A and E4.
 20. The method of claim 12 wherein the encapsidatedfully-deleted adenovirus-based gene transfer vector is replicationdeficient.